Modified Human Plasma Polypeptide or Fc Scaffolds and Their Uses

ABSTRACT

Modified human plasma polypeptides or Fc and uses thereof are provided.

FIELD OF THE INVENTION

This invention relates to human plasma polypeptides or Fc moleculesmodified to comprise at least one non-naturally-encoded amino acid.

BACKGROUND OF THE INVENTION

Human blood plasma is comprised of a variety of proteins that carry outa variety of functions. The protein components of blood plasma are afocus of intense research. See, for example, Anderson et al., Molecular& Cellular Proteomics, 3.4:311-326 (2004); and Ping et al, Proteomics,5:3506-3519 (2005) for a description of the known protein components ofhuman blood plasma. The most common protein found in human blood plasmais albumin.

Human albumin, also referred to as serum albumin, is a multifunctionalprotein found in blood plasma. It is an important factor in theregulation of plasma volume and tissue fluid balance through itscontribution to the colloid osmotic pressure of plasma. Albumin alsofunctions as a carrier for other molecules found in the bloodstream.Albumin normally constitutes 50-60% of plasma proteins and because ofits relatively low molecular weight (66,500 Daltons), exerts 80-85% ofthe colloidal osmotic pressure of the blood. Albumin regulatestransvascular fluid flux and hence, intra and extravascular fluidvolumes, and transports lipid and lipid-soluble substances. Albuminsolutions are frequently used for plasma volume expansion andmaintenance of cardiac output in the treatment of certain types of shockor impending shock including those resulting from burns, surgery,hemorrhage, or other trauma or conditions in which a circulatory volumedeficit is present.

Albumin has a blood circulation half-life of approximately two weeks andis designed by nature to carry other molecules such as lipids, peptides,and other proteins. A hydrophobic binding pocket and a free thiolcysteine residue (Cys34) are features that enable this function. Due toits low pKa (approx. 7) Cys34 is one of the more reactive thiol groupsappearing in human plasma. The Cys34 of albumin also accounts for themajor fraction of thiol concentration in blood plasma (over 80%) (Kratzet al., J. Med. Chem., 45(25):5523-33 (2002)). The ability of albuminthrough its reactive thiol to act as a carrier has been utilized fortherapeutic purposes. For example, attachment of drugs to albumin toimprove the pharmacological properties of the drugs has been described(Kremer et al., Anticancer Drugs, 13:(6):615-23 (2002); Kratz et al., J.Drug Target., 8(5):305-18 (2000); Kratz et al., J. Med. Chem.,45(25):5523-33 (2002); Tanaka et al., Bioconjug. Chem., 2(4):261-9(1991); Dosio et al., J. Control. Release, 76(1-2):107-17 (2001); Dingset al., Cancer Lett., 194(1):55-66 (2003); Wunder et al., J. Immunol.,170(9):4793-801 (2003); Christie et al., Biochem. Pharmacol.,36(20):3379-85 (1987)). The attachment of peptide and proteintherapeutics to albumin has also been described (Holmes et al.,Bioconjug. Chem., 11 (4):439-44 (2000), Leger et al., Bioorg. Med. Chem.Lett., 13(20):3571-5 (2003); Paige et al., Pharm. Res., 12(12):1883-8(1995)). Conjugates of albumin and interferon-alpha (Albuferon™) and ofalbumin and human growth hormone (Albutropin™) and of albumin andinterleukin-2 (Albuleukin™) have also been made. The art also describesthe use of standard recombinant molecular biology techniques to generatean albumin-protein fusion (U.S. Pat. No. 6,548,653, which isincorporated by reference herein). All but the latter conjugates withalbumin involve ex vivo conjugate formation with an exogenous albumin.Potential drawbacks to using exogenous sources of albumin arecontamination or an immunogenic response.

In vivo attachment of therapeutic agents to albumin has also beendescribed, where, for example, a selected peptide is modified prior toadministration to allow albumin to bind to the peptide. This approach isdescribed using dipeptidyl peptidase IV-resistantglucagon-like-peptide-1 (GLP-1) analogs (Kim et al., Diabetes,52(3):751-9 (2003)). A specific linker([2-[2-[2-maleimido-propionamido-(ethoxy)-ethoxy}-acetamide) wasattached to an added carboxyl-terminal lysine on the peptide to enable acysteine residue of albumin to bind with the peptide. Others haveinvestigated attaching specific tags to peptides or proteins in order toincrease their binding to albumin in vivo (Koehler et al., Bioorg Med.Chem. Lett., 12(20):2883-6 (2002); Dennis et al., J. Biol. Chem.,277(38):35035-35043 (2002)); Smith et al., Bioconjug. Chem., 12:750-756(2001)). A similar approach has been used with small molecule drugs,where a derivative of the drug was designed specifically to have theability to bind with a cysteine residue of albumin. For example, thispro-drug strategy has been used for doxorubicin derivatives where thedoxorubicin derivative is bound to endogenous albumin at its cysteineresidue at position 34 (Cys34; Kratz et al., J Med. Chem., 45(25):5523-33 (2002)). The in vivo attachment of a therapeutic agent toalbumin has the advantage, relative to the ex vivo approach describedabove, in that endogenous albumin is used, thus obviating problemsassociated with contamination or an immunogenic response to theexogenous albumin. Yet, the prior art approach of in vivo formation ofdrug conjugates with endogenous albumin involves a permanent covalentlinkage between the drug and the albumin. To the extent the linkage iscleavable or reversible, the drug or peptide released from the conjugateis in a modified form of the original compound.

Human serum albumin has been expressed in yeast host cells includingSaccharomyces cerevisiae (Etcheverry et al., (1986) BioTechnology 8:726,and EPA 123 544), Pichia pastoris (EPA 344 459), and Kluyveromyces(Fleer et al., (1991) BioTechnology 9:968-975), and in E. coli (Latta etal., (1987) BioTechnology 5:1309-1314), which are incorporated byreference herein.

A naturally produced antibody (Ab) is a tetrameric structure consistingof two identical immunoglobulin (Ig) heavy chains and two identicallight chains. Immunoglobulins are molecules containing polypeptidechains held together by disulfide bonds, typically having two lightchains and two heavy chains. In each chain, one domain (V) has avariable amino acid sequence depending on the antibody specificity ofthe molecule. The other domains (C) have a rather constant sequencecommon to molecules of the same class.

The heavy and light chains of an Ab consist of different domains. Eachlight chain has one variable domain (VL) and one constant domain (CL),while each heavy chain has one variable domain (VH) and three or fourconstant domains (CH). Each domain, consisting of about 110 amino acidresidues, is folded into a characteristic β-sandwich structure formedfrom two β-sheets packed against each other, the immunoglobulin fold.The VL domains each have three complementarity determining regions(CDR1-3) and the VH domains each have up to four complimentaritydetermining regions (CDR1-4), that are loops, or turns, connectingβ-strands at one end of the domains. The variable regions of both thelight and heavy chains generally contribute to antigen specificity,although the contribution of the individual chains to specificity is notnecessarily equal. Antibody molecules have evolved to bind to a largenumber of molecules by using randomized CDR loops.

Functional substructures of Abs can be prepared by proteolysis and byrecombinant methods. They include the Fab fragment, which comprises theVH-CH1 domains of the heavy chain and the VL-CL1 domains of the lightchain joined by a single interchain disulfide bond, and the Fv fragment,which comprises only the VH and VL domains, and the Fc portion whichcomprises the non-antigen binding region of the molecule. In some cases,a single VH domain retains significant affinity for antigen (Ward etal., 1989, Nature 341, 554-546). It has also been shown that a certainmonomeric κ light chain will specifically bind to its antigen. (L. Masatet al., 1994, PNAS 91:893-896). Separated light or heavy chains havesometimes been found to retain some antigen-binding activity as well(Ward et al., 1989, Nature 341, 554-546).

Another functional substructure is a single chain Fv (scFv), comprisedof the variable regions of the immunoglobulin heavy and light chain,covalently connected by a peptide linker (S-z Hu et al., 1996, CancerResearch, 56, 3055-3061). These small (Mr 25,000) proteins generallyretain specificity and affinity for antigen in a single polypeptide andcan provide a convenient building block for larger, antigen-specificmolecules. The short half-life of scFvs in the circulation limits theirtherapeutic utility in many cases.

A small protein scaffold called a “minibody” was designed using a partof the Ig VH domain as the template (Pessi et al., 1993, Nature 362,367-369). Minibodies with high affinity (dissociation constant (K_(d))about 10⁻⁷ M) to interleukin-6 were identified by randomizing loopscorresponding to CDR1 and CDR2 of VH and then selecting mutants usingthe phage display method (Martin et al., 1994, EMBO J. 13, 5303-5309).

Camels often lack variable light chain domains when IgG-like materialfrom their serum is analyzed, suggesting that sufficient antibodyspecificity and affinity can be derived from VH domains (three or fourCDR loops) alone. “Camelized” VH domains with high affinity have beenmade, and high specificity can be generated by randomizing only theCDR3.

An alternative to the “minibody” is the “diabody.” Diabodies are smallbivalent and bispecific antibody fragments, having two antigen-bindingsites. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) on the samepolypeptide chain (V_(H)-V_(L)). Diabodies are similar in size to theFab fragment. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or “diabodies,”are bivalent and bispecific. See, P. Holliger et al., PNAS 90:6444-6448(1993).

An antibody fragment includes any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like (Maynard & Georgiou, 2000, Annu.Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.9:395-402).

CDR peptides and organic CDR mimetics have been made (Dougall et al.,1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, typicallycyclic, peptides which correspond to the amino acid sequences of CDRloops of antibodies. CDR loops are responsible for antibody-antigeninteractions. CDR peptides and organic CDR mimetics have been shown toretain some binding affinity (Smyth & von Itzstein, 1994, J. Am. Chem.Soc. 116, 2725-2733). Mouse CDRs have been grafted onto the human Igframework without the loss of affinity (Jones et al., 1986, Nature 321,522-525; Riechmann et al., 1988).

In the body, specific Abs are selected and amplified from a largelibrary (affinity maturation). The processes can be reproduced in vitrousing combinatorial library technologies. The successful display of Abfragments on the surface of bacteriophage has made it possible togenerate and screen a vast number of CDR mutations (McCafferty et al.,1990, Nature 348, 552-554; Barbas et al., 1991, Proc. Natl. Acad. Sci.USA 88,7978-7982; Winter et al., 1994, Annu. Rev. Immunol. 12, 433-455).An increasing number of Fabs and Fvs (and their derivatives) areproduced by this technique. The combinatorial technique can be combinedwith Ab mimics.

A number of protein domains that could potentially serve as proteinscaffolds have been expressed as fusions with phage capsid proteins.Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994).Several of these protein domains have already been used as scaffolds fordisplaying random peptide sequences, including bovine pancreatic trypsininhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growthhormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturiniet al., Protein Peptide Letters 1:70-75 (1994)), and the IgG bindingdomain of Streptococcus (O'Neil et al., Techniques in Protein ChemistryV (Crabb, L,. ed.) pp. 517-524, Academic Press, San Diego (1994)). Thesescaffolds have displayed a single randomized loop or region. Tendamistathas been used as a presentation scaffold on the filamentous phage M13(McConnell and Hoess, 1995, J. Mol. Biol. 250:460-470).

The Fc portion of an immunoglobulin, includes but is not limited to, anantibody fragment which is obtained by removing the two antigen bindingregions (the Fab fragments) from the antibody. One way to remove the Fabfragments is to digest the immunoglobulin with papain protease. Thus,the Fc portion is formed from approximately equal sized fragments of theconstant region from both heavy chains, which associate throughnon-covalent interactions and disulfide bonds. The Fc portion caninclude the hinge regions and extend through the CH2 and CH3 domains tothe C-terminus of the antibody. Representative hinge regions for humanand mouse immunoglobulins can be found in Antibody Engineering, APractical Guide, Borrebaeck, C. A. K., ed., W. H. Freeman and Co., 1992,the teachings of which are herein incorporated by reference. The Fcportion can further include one or more glycosylation sites. The aminoacid sequences of numerous representative Fc proteins containing a hingeregion, CH2 and CH3 domains, and one N-glycosylation site are well knownin the art.

There are five types of human immunoglobulin Fc regions with differenteffector functions and pharmacokinetic properties: IgG, IgA, IgM, IgD,and IgE. IgG is the most abundant immunoglobulin in serum. IgG also hasthe longest half-life in serum of any immunoglobulin (23 days). Unlikeother immunoglobulins, IgG is efficiently recirculated following bindingto an Fc receptor. There are four IgG subclasses G1, G2, G3, and G4,each of which has different effector functions. G1, G2, and G3 can bindC1q and fix complement while G4 cannot. Even though G3 is able to bindClq more efficiently than G1, G1 is more effective at mediatingcomplement-directed cell lysis. G2 fixes complement very inefficiently.The C 1q binding site in IgG is located at the carboxy terminal regionof the CH2 domain.

All IgG subclasses are capable of binding to Fc receptors (CD16, CD32,CD64) with GI and G3 being more effective than G2 and G4. The Fcreceptor binding region of IgG is formed by residues located in both thehinge and the carboxy terminal regions of the CH2 domain.

IgA can exist both in a monomeric and dimeric form held together by aJ-chain. IgA is the second most abundant Ig in serum, but it has ahalf-life of only 6 days. IgA has three effector functions. It binds toan IgA specific receptor on macrophages and eosinophils, which drivesphagocytosis and degranulation, respectively. It can also fix complementvia an unknown alternative pathway.

IgM is expressed as either a pentamer or a hexamer, both of which areheld together by a J-chain. IgM has a serum half-life of 5 days. Itbinds weakly to C1q via a binding site located in its CH3 domain. IgDhas a half-life of 3 days in serum. It is unclear what effectorfunctions are attributable to this Ig. IgE is a monomeric Ig and has aserum half-life of 2.5 days. IgE binds to two Fc receptors which drivesdegranulation and results in the release of proinflammatory agents.

Polypeptides of the present invention may contain any of the isotypesdescribed above or may contain mutated Fc regions wherein the complementand/or Fc receptor binding functions have been altered, modified, orremoved. Polypeptides of the present invention may contain any of theisotypes described above or may contain mutated Fc regions wherein theeffector function has been altered, modified, or removed. Thus, thepolypeptides of the present invention may contain the entire Fc portionof an immunoglobulin, fragments of the Fc portion of an immunoglobulin,or analogs thereof.

Polypeptides of the present invention can consist of single chainproteins or as multi-chain polypeptides. Two or more Fc proteins can beproduced such that they interact through disulfide bonds that naturallyform between Fc regions. These multimers can be homogeneous with respectto a conjugated molecule or they may contain different conjugatedmolecules at the N-terminus of the Fc portion of the fusion protein.

A Fc or Fc-like region may serve to prolong the in vivo plasma half-lifeof a compound fused to it. Since the Fc region of IgG produced byproteolysis has the same in vivo half-life as the intact IgG moleculeand Fab fragments are rapidly degraded, it is believed that the relevantsequence for prolonging half-life reside in the CH2 and/or CH3 domains.Further, it has been shown in the literature that the catabolic rates ofIgG variants that do not bind the high-affinity Fc receptor or C1q areindistinguishable from the rate of clearance of the parent wild-typeantibody, indicating that the catabolic site is distinct from the sitesinvolved in Fc receptor or C1q binding. [Wawrzynczak et al., (1992)Molecular Immunology 29:221]. Site-directed mutagenesis studies using amurine IgG1 Fc region suggested that the site of the IgG1 Fc region thatcontrols the catabolic rate is located at the CH2-CH3 domain interface.Fc regions can be modified at the catabolic site to optimize thehalf-life of the fusion proteins. The Fc region used for the fusionproteins of the present invention may be derived from an IgG 1 or anIgG4 Fc region, and may contain both the CH2 and CH3 regions includingthe hinge region.

Chimeric molecules comprising Fc and one or more other moleculesincluding, but not limited to, a polypeptide may be generated. Thechimeric molecule can contain specific regions or fragments of Fc andthe other molecule(s). Any such fragments can be prepared from theproteins by standard biochemical methods, or by expressing apolynucleotide encoding the fragment. A polypeptide, or a fragmentthereof, can be produced as a fusion protein comprising human serumalbumin (HSA), Fc, or a portion thereof. Fusions may be created byfusion of a polypeptide with a) the Fc portion of an immunoglobulin; b)an analog of the Fc portion of an immunoglobulin; and c) fragments ofthe Fc portion of an immunoglobulin.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003), Drabkin etal., (1996) Mol.Cell.Biol., 16:907) and in mammalian cells (Sakamoto etal., (2002) Nucleic Acids Res. 30:4692), which has enabled theincorporation of non-genetically encoded amino acids to proteins invivo. A number of new amino acids with novel chemical, physical orbiological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U. S. A.99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc.124:9026-9027), keto amino acids, heavy atom containing amino acids, andglycosylated amino acids have been incorporated efficiently and withhigh fidelity into proteins in E. coli and in yeast in response to theamber codon, TAG, using this methodology. See, e.g., J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137; J. W. Chin,et al., (2002), PNAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. Comm. 1:1-11. All references areincorporated by reference in their entirety. These studies havedemonstrated that it is possible to selectively and routinely introducechemical functional groups, such as ketone groups, alkyne groups andazide moieties, that are not found in proteins, that are chemicallyinert to all of the functional groups found in the 20 common,genetically-encoded amino acids and that may be used to reactefficiently and selectively to form stable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SHof cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups such as carbolyl, alkyne, and azide moieties describedherein are known to be inert to the functional groups found in the 20common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages.

SUMMARY OF THE INVENTION

This invention provides human plasma protein (hPP) family members,including but not limited to, plasma proteins that function as carriersof other molecules. The hPP's that may be suitable for use in thepresent invention include but are not limited to those proteins listedin the following publications, which are incorporated by referenceherein in their entirety: Anderson et al., Molecular & CellularProteomics, 3.4:311-326 (2004); and Ping et al, Proteomics, 5:3506-3519(2005). Some of the known hPP's include but are not limited to,α1-antichymotrypsin, antitrypsin, α1 -antitrypsin, pre-ablumin, humanalbumin (human serum albumin), α1-lipoprotein, A-gamma globulin,α2-macroglobulin, α1-microglobulin, α2-microglobulin, β2-microglobulin,Bence Jones protein, bile secretory component, compliment protein 3,cholesteryl ester transfer protein, fatty acid binding protein,ferritin, ferritin H chain, fibrinogen, gastric inhibitory peptide,globulins, haptoglobulin, hemoglobin, hemoglobin A, hemoglobin A1C,hemoglobin F, glycated hemoglobin, pan hemoglobin, lactoferrin, lipase,lysozyme, mutY, myoglobin, cardiac myoglobin, orosmucoid, rheumatoidfactor, secretin, serotonin, thyroglobulin, thyroxine, thyroxine bindingglobulin, triiodothyronine, transferring, vitamin D binding protein, andvariant forms thereof, comprising one or more non-naturally encodedamino acids. This invention also provides human Fc (hFc) comprising oneor more non-naturally encoded amino acids.

In some embodiments, the hPP or hFc comprises one or morepost-translational modifications. In some embodiments, the hPP or hFc islinked to a linker, polymer, or biologically active molecule. In someembodiments, the hPP or hFc is linked to a bifunctional ormultifunctional polymer, bifunctional or multifunctional linker, or atleast one additional biologically active molecule.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is directly bonded to the water soluble polymer. Insome embodiments, the poly(ethylene glycol) molecule is a bifunctionalor multifunctional polymer. In some embodiments, the bifunctional ormultifunctional polymer is linked to a second polypeptide. In someembodiments, the second polypeptide is a biologically active molecule.

In some embodiments, the hPP or hFc comprises at least two amino acidslinked to a water soluble polymer, a linker, or a biologically activemolecule. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, the hPP comprising one or more non-naturallyencoded amino acids is human albumin (hA), which is also known in theart as human serum albumin. The hA may be substituted with one or morenon-naturally encoded amino acids at one or more of the 582 positions ofthe polypeptide sequence, including but not limited to one or more ofthe following positions: before position 1 (i.e. at the N-terminus), 17,34, 55, 56, 58, 60, 81, 82, 86, 92, 94, 111, 114, 116, 119, 129, 170,172, 173, 276, 277, 280, 297, 300, 301, 313, 317, 321, 362, 363, 364,365, 368, 375, 397, 439, 442, 495, 496, 498, 500, 501, 505, 515, 538,541, 542, 560, 562, 564, 574, 581, and after position 582 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1).

hFc may be substituted with one or more non-naturally encoded aminoacids at one or more of the positions of the polypeptide sequence,including but not limited to, before position 1 (i.e. at the N-terminus)and at the N terminus (SEQ ID NO: 22).

In some embodiments, the hPP or hFc comprises a substitution, additionor deletion that modulates affinity of the hPP or hFc for an hPP or hFcreceptor or binding partner, including but not limited to, a protein,lipid, saccharide, polypeptide, small molecule, or nucleic acid. In someembodiments, the hPP or hFc comprises a substitution, addition, ordeletion that increases the stability of the hPP or hFc when comparedwith the stability of the corresponding hPP or hFc without thesubstitution, addition, or deletion. In some embodiments, the hPP or hFccomprises a substitution, addition, or deletion that modulates theimmunogenicity of the hPP or hFc when compared with the immunogenicityof the corresponding hPP or hFc without the substitution, addition, ordeletion. In some embodiments, the hPP or hFc comprises a substitution,addition, or deletion that modulates serum half-life or circulation timeof the hPP or hFc when compared with the serum half-life or circulationtime of the corresponding hPP or hFc without the substitution, addition,or deletion.

In some embodiments, the hPP or hFc comprises a substitution, addition,or deletion that increases the aqueous solubility of the hPP or hFc whencompared to aqueous solubility of the corresponding hPP or hFc withoutthe substitution, addition, or deletion. In some embodiments, the hPP orhFc comprises a substitution, addition, or deletion that increases thesolubility of the hPP or hFc produced in a host cell when compared tothe solubility of the corresponding hPP or hFc without the substitution,addition, or deletion. In some embodiments, the hPP or hFc comprises asubstitution, addition, or deletion that increases the expression of thehPP or hFc in a host cell or increases synthesis in vitro when comparedto the expression or synthesis of the corresponding hPP or hFc withoutthe substitution, addition, or deletion. The hPP or hFc comprising thissubstitution retains agonist activity and retains or improves expressionlevels in a host cell. In some embodiments, the hPP or hFc comprises asubstitution, addition, or deletion that increases protease resistanceof the hPP or hFc when compared to the protease resistance of thecorresponding hPP or hFc without the substitution, addition, ordeletion.

In some embodiments the amino acid substitutions in the hPP or hFc maybe with naturally occurring or non-naturally occurring amino acids,provided that at least one substitution is with a non-naturally encodedamino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, an alkyne group,an aniline amino group, or a saccaride moiety.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to SEQ ID NO:2, 20 or 23 wherein the polynucleotide comprises at least one selectorcodon. In some embodiments, the selector codon is selected from thegroup consisting of an amber codon, ochre codon, opal codon, a uniquecodon, a rare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making an hPP or hFclinked to a water soluble polymer. In some embodiments, the methodcomprises contacting an isolated hPP comprising a non-naturally encodedamino acid with a water soluble polymer comprising a moiety that reactswith the non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid incorporated into the hPP or hFc isreactive toward a water soluble polymer that is otherwise unreactivetoward any of the 20 common amino acids. In some embodiments, thenon-naturally encoded amino acid incorporated into the hPP or hFc isreactive toward a linker, polymer, or biologically active molecule thatis otherwise unreactive toward any of the 20 common amino acids.

In some embodiments, the hPP or hFc linked to the water soluble polymeris made by reacting an hPP or hFc comprising a carbonyl-containing aminoacid with a poly(ethylene glycol) molecule comprising an aminooxy,hydrazine, hydrazide or semicarbazide group. In some embodiments, theaminooxy, hydrazine, hydrazide or semicarbazide group is linked to thepoly(ethylene glycol) molecule through an amide linkage.

In some embodiments, the hPP or hFc linked to the water soluble polymeris made by reacting a poly(ethylene glycol) molecule comprising acarbonyl group with a polypeptide comprising a non-naturally encodedamino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group.

In some embodiments, the hPP or hFc linked to the water soluble polymeris made by reacting an hPP or hFc comprising an alkyne-containing aminoacid with a poly(ethylene glycol) molecule comprising an azide moiety.In some embodiments, the azide or alkyne group is linked to thepoly(ethylene glycol) molecule through an amide linkage.

In some embodiments, the hPP or hFc linked to the water soluble polymeris made by reacting an hPP or hFc comprising an azide-containing aminoacid with a poly(ethylene glycol) molecule comprising an alkyne moiety.In some embodiments, the azide or alkyne group is linked to thepoly(ethylene glycol) molecule through an amide linkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the hPPcomprises a polyalkylene gl ycol moiety. I n some embodiments, thenon-naturally encoded amino acid residue incorporated into the hPP orhFc comprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine, a semicarbazide group, an azide group, or an alkyne group. Insome embodiments, the non-naturally encoded amino acid residueincorporated into the hPP or hFc comprises a carbonyl moiety and thewater soluble polymer comprises an aminooxy, hydrazide, hydrazine, orsemicarbazide moiety. In some embodiments, the non-naturally encodedamino acid residue incorporated into the hPP or hFc comprises an alkynemoiety and the water soluble polymer comprises an azide moiety. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto the hPP or hFc comprises an azide moiety and the water solublepolymer comprises an alkyne moiety.

The present invention also provides compositions comprising an hPP orhFc comprising a non-naturally encoded amino acid and a pharmaceuticallyacceptable carrier. In some embodiments, the non-naturally encoded aminoacid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the hPP or hFc comprising a selector codon. In someembodiments, the cells comprise an orthogonal RNA synthetase and/or anorthogonal tRNA for substituting a non-naturally encoded amino acid intothe hPP or hFc.

The present invention also provides methods of making an hPP or hFccomprising a non-naturally encoded amino acid. In some embodiments, themethods comprise culturing cells comprising a polynucleotide orpolynucleotides encoding an hPP or hFc, an orthogonal RNA synthetaseand/or an orthogonal tRNA under conditions to permit expression of thehPP or hFc; and purifying the hPP or hFc from the cells and/or culturemedium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of hPP or hFc. Thepresent invention also provides methods of modulating immunogenicity ofhPP or hFc. In some embodiments, the methods comprise substituting anon-naturally encoded amino acid for any one or more amino acids innaturally occurring hPPs or hFc and/or linking the hPP or hFc to alinker, a polymer, a water soluble polymer, or a biologically activemolecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of an hPP or hFcmolecule of the present invention. In some embodiments, the methodscomprise administering to the patient a therapeutically-effective amountof a pharmaceutical composition comprising an hPP or hFc comprising anon-naturally-encoded amino acid and a pharmaceutically acceptablecarrier. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer.

The present invention also provides hPP comprising an amino acidsequence shown in SEQ ID NO: 1, or any other hPP polypeptide sequence,except that at least one amino acid is substituted by a non-naturallyencoded amino acid. The present invention also provides hFc comprisingan amino acid sequence shown in SEQ ID NO: 22, or any other hFcpolypeptide sequence, except that at least one amino acid is substitutedby a non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.In some embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the non-naturally encoded aminoacid comprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an hPP comprisingthe sequence shown in SEQ ID NO: 1, or any other hPP polypeptidesequence, wherein at least one amino acid is substituted by anon-naturally encoded amino acid. The present invention also providespharmaceutical compositions comprising a pharmaceutically acceptablecarrier and an hPP comprising the sequence shown in SEQ ID NO: 22 or anyother hFc polypeptide sequence, wherein at least one amino acid issubstituted by a non-naturally encoded amino acid. In some embodiments,the non-naturally encoded amino acid comprises a saccharide moiety. Insome embodiments, the water soluble polymer is linked to the polypeptidevia a saccharide moiety. In some embodiments, a linker, polymer, orbiologically active molecule is linked to the hPP or hFc via asaccharide moiety.

The present invention also provides an hPP or hFc comprising a watersoluble polymer linked by a covalent bond to the hPP or hFc at a singleamino acid. In some embodiments, the water soluble polymer comprises apoly(ethylene glycol) moiety. In some embodiments, the amino acidcovalently linked to the water soluble polymer is a non-naturallyencoded amino acid present in the polypeptide.

The present invention provides an hPP or hFc comprising at least onelinker, polymer, or biologically active molecule, wherein said linker,polymer, or biologically active molecule is attached to the polypeptidethrough a functional group of a non-naturally encoded amino acidribosomally incorporated into the polypeptide. In some embodiments, thepolypeptide is monoPEGylated. The present invention also provides an hPPor hFc comprising a linker, polymer, or biologically active moleculethat is attached to one or more non-naturally encoded amino acid whereinsaid non-naturally encoded amino acid is ribosomally incorporated intothe polypeptide at pre-selected sites.

In another embodiment, conjugation of the hPP or hFc comprising one ormore non-naturally occurring amino acids to another molecule, includingbut not limited to PEG, provides substantially purified hPP or hFc dueto the unique chemical reaction utilized for conjugation to thenon-natural amino acid. In another embodiment, one or more non-naturallyencoded amino acids are incorporated into the amino acid sequence of anhPP or hFc provides advantages for purification of the hPP or hFcutilizing the functional group of the non-naturally encoded amino acid.Conjugation of hPP or hFc comprising one or more non-naturally encodedamino acids to another molecule, such as PEG, may be performed withother purification techniques performed prior to or following theconjugation step to provide substantially pure hPP or hFc. In anotherembodiment substitution of a non-naturally encoded amino acid into theamino acid sequence of an hPP or hFc modulates the pKa of thepolypeptide, which in turn modulates the conjugation reactionconditions, rate or efficiency when conjugating the hPP or hFc to othermolecules. In some embodiments the hPP or hFc comprising thenon-naturally encoded amino acid exhibits modulated bindingcharacteristics, such as increased or decreased binding strength, whencontacted with a binding partner. In some embodiments the hPP or hFccomprising the non-naturally encoded amino acid exhibits modulatedtissue binding or tissue distribution characteristics when compared tothe same hPP or hFc lacking the non-naturally encoded amino acid. Insome embodiments the hPP or hFc comprising the non-naturally encodedamino acid has modulated stability properties when the hPP or hFc isformulated for pharmaceutical uses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, and 1B—The amino acid sequence of mature human albumin isshown in 1A, and the nucleotide sequence encoding human albumin is shownin 1B.

FIG. 2—The average Cx values for hA amino acids is shown.

FIG. 3—A model of hA and certain amino acids positions are shown.

FIG. 4—A table of selected amino acid positions in hA for substitutionwith non-naturally encoded amino acids is shown.

FIG. 5—The expression of recombinant human albumin in yeast host cellsis shown by coomassie stained polyacrylamide gel electrophoresis.

FIG. 6—The expression of hA containing a non-naturally encoded aminoacid in the polypeptide sequence is shown by coomassie stainedpolyacrylamide gel electrophoresis.

FIG. 7, Panel A—Reduced samples of purified Fc (WT) andD1pAF-substituted Fc (D1pAF) incubated in the presence (+) or absence(−) of 5K amino-oxy poly(ethylene)-glycol (PEG) were analyzed bySDS-PAGE.

FIG. 7, Panel B—Non-reduced samples of purified Fc (WT) andD1pAF-substituted Fc (D1pAF) incubated in the presence (+) or absence(−) of 5K amino-oxy poly(ethylene)-glycol (PEG) were analyzed bySDS-PAGE.

FIG. 8A—The polynucleotide sequence of a wild type Fc is shown.

FIG. 8B—The polypeptide sequence of a wild type Fc is shown.

FIG. 8C—The polypeptide sequence of a mature Fc is shown.

FIG. 8D—The polynucleotide sequence encoding a mature Fc is shown.

FIG. 9A—The polynucleotide sequence of a tRNA is shown.

FIG. 9B—The polynucleotide sequence encoding a aminoacyl-tRNA synthetaseis shown.

FIG. 10A—The incorporation of non-natural amino acid into hA is shown ina coomassie stained polyacrylamide gel.

FIG. 10B—The incorporation of non-natural amino acids into hA is shownin a Western blot.

FIG. 10C—The incorporation of non-natural amino acids into hA is shownin a Western blot.

FIG. 10C—The incorporation of non-natural amino acids into hA is shownin a Western blot.

FIG. 11—The production of PEGylated hA is shown by Western blot.

FIG. 12 Panel A and FIG. 12 Panel B—Peptide mapping results for hAcomprising a non-natural amino acid, and wild type hA, respectively, areshown.

FIG. 13A—Mass spectroscopy results of hA comprising non-natural aminoacid (pAF, or para acetyl phenylalanine) is shown.

FIG. 13B—The predicted ion masses for the amino acids of hA, includingnon-natural amino acid pAF (para acetyl phenylalanine) is shown.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to an “hPP” or “hA” or “hFc” isa reference to one or more such proteins and includes equivalentsthereof known to those of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to an “hPP” or “hA” or hFcpolypeptide that may be substantially or essentially free of componentsthat normally accompany or interact with the protein as found in itsnaturally occurring environment, i.e. a native cell, or host cell in thecase of recombinantly produced an “hPP” or “hA” or “hFc” polypeptide. An“hPP” or “hA” or “hFc” polypeptide that may be substantially free ofcellular material includes preparations of protein having less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2%, or less than about 1% (by dry weight)of contaminating protein. When an “hPP” or “hA” or “hFc” polypeptide orvariant thereof is recombinantly produced by the host cells, the proteinmay be present at about 30%, about 25%, about 20%, about 15%, about 10%,about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dryweight of the cells. When an “hPP” or “hA” or “hFc” polypeptide orvariant thereof is recombinantly produced by the host cells, the proteinmay be present in the culture medium at about 5 g/L, about 4 g/L, about3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/Lor less of the dry weight of the cells. Thus, “substantially purified”“hPP” or “hA” or “hFc” polypeptide as produced by the methods of thepresent invention may have a purity level of at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, specifically, a purity level of at least about 75%,80%, 85%, and more specifically, a purity level of at least about 90%, apurity level of at least about 95%, a purity level of at least about 99%or greater as determined by appropriate methods such as SDS/PAGEanalysis, RP-HPLC, SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into which an“hPP” or “hA” or “hFc” polypeptide has been secreted, including mediumeither before or after a proliferation step. The term also may encompassbuffers or reagents that contain host cell lysates, such as in the casewhere an “hPP” or “hA” or “hFc”polypeptide is produced intracellularlyand the host cells are lysed or disrupted to release the “hPP” or “hA”or “hFc”polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy- 1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

As used herein, “human plasma protein or polypeptide” or “hPP” includesthose polypeptides and proteins that are found in normal human bloodplasma, including hPP analogs, hPP isoforms, hPP mimetics, hPPfragments, hybrid hPP proteins, fusion proteins, oligomers andmultimers, homologues, glycosylation pattern variants, variants, splicevariants, and muteins, thereof, regardless of the biological activity ofsame, and further regardless of the method of synthesis or manufacturethereof including, but not limited to, recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), invitro, in vivo, by microinjection of nucleic acid molecules, synthetic,transgenic, and gene activated methods. A variety of hPP's are known inthe art and can be found in Anderson et al., Molecular & CellularProteomics, 3.4:311-326 (2004); and Ping et al, Proteomics, 5:3506-3519(2005), which are incorporated by reference herein. The hPP useful inthe present invention may include but are not limited toα1-antichymotrypsin, antitrypsin, α1-antitrypsin, pre-ablumin, humanalbumin (human serum albumin), α1-lipoprotein, A-gamma globulin,α2-macroglobulin, α1-microglobulin, α2-microglobulin, β2-microglobulin,Bence Jones protein, bile secretory component, compliment protein 3,cholesteryl ester transfer protein, fatty acid binding protein,ferritin, ferritin H chain, fibrinogen, gastric inhibitory peptide,globulins, haptoglobulin, hemoglobin, hemoglobin A, hemoglobin A 1 C,hemoglobin F, glycated hemoglobin, pan hemoglobin, lactoferrin, lipase,lysozyme, mutY, myoglobin, cardiac myoglobin, orosmucoid, rheumatoidfactor, secretin, serotonin, thyroglobulin, thyroxine, thyroxine bindingglobulin, triiodothyronine, transferring, vitamin D binding protein, andvariant forms thereof.

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin (“hA”) or fragmentsthereof especially the mature form of human albumin as shown in SEQ IDNO:1, or albumin from other vertebrates such as bovine, porcine, equine,canine, feline, or avian, or fragments thereof, or analogs or variantsof these molecules or fragments thereof. The amino acid sequence and thenucleotide sequence of hA are known in the art and disclosed, forexample, in U.S. Pat. Nos. 5,879,907; 5,756,313; 5,707,828; 5,986,062;5,521,287; 5,612,197; 5,440,-18; 5,759,819; and 5,648,243, which areincorporated by reference herein.

In some embodiments, the human serum albumin protein used in the presentinvention contains one or both of the following sets of point mutationswith reference to SEQ ID NO:1: Leu-407 to Ala, Leu-408 to Val, Val-409to Ala, and Arg-410 to Ala; or Arg-410 to Ala, Lys-413 to Gln, andLys-414 to Gln (see, e.g., International Patent Publication No.WO95/23857, hereby incorporated by reference herein in its entirety). Inother embodiments, albumin fusion proteins of the present invention thatcontain one or both of above-described sets of point mutations haveimproved stability/resistance to yeast Yap3p proteolytic cleavage,allowing increased production of recombinant albumin fusion proteinsexpressed in yeast host cells.

As used herein, a portion of hA sufficient to prolong the therapeuticactivity, circulation time, or shelf-life of a therapeutic productrefers to a portion of hA sufficient in length or structure to stabilizeor prolong the therapeutic activity of the protein. The albumin portionof the proteins may comprise the full length of the hA sequence asdescribed herein, or may include one or more fragments thereof that arecapable of providing the desired activity. Such hA fragments may be of10 or more amino acids in length or may include about 15, 20, 25, 30,50, 100 or more contiguous amino acids from the hA sequence or mayinclude part or all of specific domains of hA. For example, one or morefragments of hA spanning the first two immunoglobulin-like domains maybe used. Examples of truncated forms of hA may be found in U.S. Pat. No.5,380,712, which is incorporated by reference herein.

The albumin portion of the albumin fusion proteins of the invention maybe a variant of normal hA. The therapeutic protein portion of thealbumin fusion proteins of the invention may also be variants of thetherapeutic proteins as described herein. The term “variants” includesinsertions, deletions and substitutions, either conservative or nonconservative, where such changes do not substantially alter one or moreof the oncotic, useful ligand-binding and non-immunogenic properties ofalbumin, or the active site, or active domain which confers thetherapeutic activities of the therapeutic proteins.

In particular, the hA proteins of the invention may include naturallyoccurring polymorphic variants of hA and fragments of hA, for examplethose fragments disclosed in EP 322 094. The albumin may be derived fromany vertebrate, especially any mammal, for example human, cow, sheep, orpig. Non-mammalian albumins include, but are not limited to, hen andsalmon. The albumin portion of the albumin fusion protein may be from adifferent animal than a molecule that may be coupled to the hA. An hAfragment or variant may also be utilized in the present invention. ThehA variant may consist of or alternatively comprise at least onecomplete structural domain of hA, for example domains 1 (amino acids1-194 of SEQ ID NO:1), 2 (amino acids 195-387 of SEQ ID NO:1), 3 (aminoacids 388-585 of SEQ ID NO:1), ½ (1-387 of SEQ ID NO:1), ⅔ (195-585 ofSEQ ID NO:1) or ⅓ (amino acids 1-194 of SEQ ID NO:1 and amino acids388-585 of SEQ ID NO:1). Each domain is itself made up of two homologoussubdomains namely 1-105, 120-194, 195-291, 316 387, 388 491 and 512 585,with flexible inter-subdomain linker regions comprising residues Lys106to Glu 119, Glu292 to Val315 and Glu492 to Ala511. Preferably, the hAportio n of an hA p rotein of the present invention comprises at leastone subdomain or domain of hA or conservative modifications thereof. Ifthe hA is based on subdomains, some or all of the adjacent linker ispreferably used to link to another molecule such as a linker, polymer,or biologically active molecule.

For the complete full-length naturally-occurring hA amino acid sequence,see SEQ ID NO: 1 herein. In some embodiments, hA polypeptides of theinvention are substantially identical to SEQ ID NO: 1. For the completenucleic acid sequence encoding hA, see SEQ ID NO: 2 herein. In someembodiments, hA polypeptides of the invention are encoded by a nucleicacid sequence substantially identical to SEQ ID NO: 2.

The term “hA” also includes the pharmaceutically acceptable salts andprodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates,biologically-active fragments, biologically active variants andstereoisomers of the naturally-occurring hA as well as agonist, mimetic,and antagonist variants of the naturally-occurring hA and polypeptidefusions thereof. Fusions comprising additional amino acids at the aminoterminus, carboxyl terminus, or both, are encompassed by the term “hApolypeptide.”

Antibodies are proteins, which exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called Complementarity DeterminingRegions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three or four CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgAl and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ and μ respectively. Of the varioushuman immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM areknown to activate complement.

In vivo, affinity maturation of antibodies is driven by antigenselection of higher affinity antibody variants which are made primarilyby somatic hypermutagenesis. A “repertoire shift” also often occurs inwhich the predominant germline genes of the secondary or tertiaryresponse are seen to differ from those of the primary or secondaryresponse.

The affinity maturation process of the immune system may be replicatedby introducing mutations into antibody genes in vitro and using affinityselection to isolate mutants with improved affinity. Such mutantantibodies can be displayed on the surface of filamentous bacteriophageor microorganisms such as yeast, and antibodies can be selected by theiraffinity for antigen or by their kinetics of dissociation (off-rate)from antigen. Hawkins et al. J. Mol. Biol. 226:889-896 (1992). CDRwalking mutagenesis has been employed to affinity mature humanantibodies which bind the human envelope glycoprotein gp120 of humanimmunodeficiency virus type 1 (HIV-1) (Barbas III et al. PNAS (USA) 91:3809-3813 (1994); and Yang et al. J. Mol. Biol. 254:392-403 (1995)); andan anti-c-erbB-2 single chain Fv fragment (Schier et al. J. Mol. Biol.263:551567 (1996)). Antibody chain shuffling and CDR mutagenesis wereused to affinity mature a high-affinity human antibody directed againstthe third hypervariable loop of HIV (Thompson et al. J. Mol. Biol.256:77-88 (1996)). Balint and Larrick Gene 137:109-118 (1993) describe acomputer-assisted oligodeoxyribonucleotide-directed scanning mutagenesiswhereby all CDRs of a variable region gene are simultaneously andthoroughly searched for improved variants. An αvβ3-specific humanizedantibody was affinity matured using an initial limited mutagenesisstrategy in which every position of all six CDRs was mutated followed bythe expression and screening of a combinatorial library including thehighest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).Phage displayed antibodies are reviewed in Chiswell and McCaffertyTIBTECH 10:80-84 (1992); and Rader and Barbas III Current Opinion inBiotech. 8:503-508 (1997). In each case where mutant antibodies withimproved affinity compared to a parent antibody are reported in theabove references, the mutant antibody has amino acid substitutions in aCDR.

By “affinity maturation” herein is meant the process of enhancing theaffinity of an antibody for its antigen. Methods for affinity maturationinclude but are not limited to computational screening methods andexperimental methods.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the antibody genes.The immunoglobulin genes include, but are not limited to, the kappa,lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Antibody herein is meant to include full-length antibodiesand antibody fragments, and include antibodies that exist naturally inany organism or are engineered (e.g. are variants).

By “antibody fragment” is meant any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab') ₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like (Maynard & Georgiou, 2000, Annu.Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.9:395-402).

By “Fc” herein is meant the portions of an antibody that are comprisedof immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3). Fc may also includeany residues which exist in the N-terminal hinge between Cγ2 and Cγ1(Cγ1). Fc may refer to this region in isolation, or this region in thecontext of an antibody or antibody fragment. Fc also includes anymodified forms of Fc, including but not limited to the native monomer,the native dimer (disulfide bond linked), modified dimers (disulfideand/or non-covalently linked), and modified monomers (i.e.,derivatives). “hFc” refers to human Fc.

By “full-length antibody” herein is meant the structure that constitutesthe natural biological form of an antibody H and/or L chain. In mostmammals, including humans and mice, this form is a tetramer and consistsof two identical pairs of two immunoglobulin chains, each pair havingone light and one heavy chain, each light chain comprisingimmunoglobulin domains V_(L) and C _(L), and each heavy chain comprisingimmunoglobulin domains V_(H), Cγ1, Cγ2, and Cγ3. In each pair, the lightand heavy chain variable regions (V_(L) and V_(H)) are togetherresponsible for binding to an antigen, and the constant regions (C_(L),Cγ1, Cγ2, and Cγ3, particularly Cγ2, and Cγ3) are responsible forantibody effector functions. In some mammals, for example in camels andllamas, full-length antibodies may consist of only two heavy chains,each heavy chain comprising immunoglobulin domains V_(H), Cγ2, and Cγ3.

By “immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains including but not limited to V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “immunoglobulin (Ig) domain” herein is meant a protein domainconsisting of a polypeptide substantially encoded by an immunoglobulingene. Ig domains include but are not limited to V_(H), Cγ1, Cγ2, Cγ3,V_(L), and C_(L).

By “variant protein sequence” as used herein is meant a protein sequencethat has one or more residues that differ in amino acid identity fromanother similar protein sequence. Said similar protein sequence may bethe natural wild type protein sequence, or another variant of the wildtype sequence. In general, a starting sequence is referred to as a“parent” sequence, and may either be a wild type or variant sequence.For example, preferred embodiments of the present invention may utilizehumanized parent sequences upon which computational analyses are done tomake variants.

By “variable region” of an antibody herein is meant a polypeptide orpolypeptides composed of the V_(H) immunoglobulin domain, the V_(L)immunoglobulin domains, or the V_(H) and V_(L) immunoglobulin domains(including variants). Variable region may refer to this or thesepolypeptides in isolation, as an Fv fragment, as a scFv fragment, asthis region in the context of a larger antibody fragment, or as thisregion in the context of a full-length antibody or an alternative,non-antibody scaffold molecule.

The present invention may be applied to antibodies obtained from a widerange of sources. The antibody may be substantially encoded by anantibody gene or antibody genes from any organism, including but notlimited to humans, mice, rats, rabbits, camels, llamas, dromedaries,monkeys, particularly mammals and particularly human and particularlymice and rats. In one embodiment, the antibody may be fully human,obtained for example from a patient or subject, by using transgenic miceor other animals (Bruggemann & Taussig, 1997, Curr. Opin. Biotechnol.8:455-458) or human antibody libraries coupled with selection methods(Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108). Theantibody may be from any source, including artificial or naturallyoccurring. For example the present invention may utilize an engineeredantibody, including but not limited to chimeric antibodies and humanizedantibodies (Clark, 2000, Immunol. Today 21:397-402) or derived from acombinatorial library. In addition, the antibody being optimized may bean engineered variant of an antibody that is substantially encoded byone or more natural antibody genes. For example, in one embodiment theantibody being optimized is an antibody that has been identified byaffinity maturation.

As used herein, “hFc” or “human Fc” or “hFc polypeptides” includes hFcanalogs, hFc isoforms, hFc mimetics, hFc fragments, hybrid hFc proteins, fusion proteins, oligomers and multimers, homologues,glycosylation pattern variants, variants, splice variants, and muteins,thereof, regardless of the biological activity of same, and furtherregardless of the method of synthesis or manufacture thereof including,but not limited to, recombinant (whether produced from cDNA, genomicDNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, bymicroinjection of nucleic acid molecules, synthetic, transgenic, andgene activated methods. A variety of hFc's are known in the art.

The term “hFc” also includes the pharmaceutically acceptable salts andprodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates,biologically-active fragments, biologically active variants andstereoisomers of hFc as well as agonist, mimetic, and antagonistvariants of the hFc and polypeptide fusions thereof. Fusions comprisingadditional amino acids at the amino terminus, carboxyl terminus, orboth, are encompassed by the term “hFc polypeptide.”

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “hPP polypeptide” or “hApolypeptide” or “hFc polypeptide” includes polypeptides conjugated to apolymer such as PEG and may be comprised of one or more additionalderivitizations of cysteine, lysine, or other residues. In addition, thehPP polypeptide or hA or hFc polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

The term “hPP polypeptide” or “hA polypeptide” or “hFc polypeptide” alsoincludes glycosylated forms, such as but not limited to, polypeptidesglycosylated at any amino acid position, N-linked or 0-linkedglycosylated forms of the polypeptide. Variants containing singlenucleotide changes are also considered as biologically active variantsof hPP polypeptide or hA polypeptide or hFc polypeptide. In addition,splice variants are also included. The term hPP polypeptide or hApolypeptide or hFc polypeptide also includes hPP or hA or hFcpolypeptide heterodimers, homodimers, heteromultimers, or homomultimersof any one or more hPP or hA or hFc polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

All references to amino acid positions in hA described herein are basedon the position in SEQ ID NO: 1, unless otherwise specified. Those ofskill in the art will appreciate that amino acid positions correspondingto positions in SEQ ID NO: 1 or any other hA sequence can be readilyidentified in any other hA molecule such as hA fusions, variants,fragments, etc. For example, sequence alignment programs such as BLASTcan be used to align and identify a particular position in a proteinthat corresponds with a position in SEQ ID NO: 1, 2, or other hAsequence. Substitutions, deletions or additions of amino acids describedherein in reference to SEQ ID NO: 1, or other hA sequence are intendedto also refer to substitutions, deletions or additions in correspondingpositions in hA fusions, variants, fragments, etc. described herein orknown in the art and are expressly encompassed by the present invention.

All references to amino acid positions in hFc described herein are basedon the position in SEQ ID NO: 22, unless otherwise specified. Those ofskill in the art will appreciate that amino acid positions correspondingto positions in SEQ ID NO: 22 or any other hFc sequence can be readilyidentified in any other hFc molecule such as hFc fusions, variants,fragments, etc. For example, sequence alignment programs such as BLASTcan be used to align and identify a particular position in a proteinthat corresponds with a position in SEQ ID NO: 20, 21, 22, 23, or otherhFc sequence. Substitutions, deletions or additions of amino acidsdescribed herein in reference to SEQ ID NO: 22, or other hFc sequenceare intended to also refer to substitutions, deletions or additions incorresponding positions in hFc fusions, variants, fragments, etc.described herein or known in the art and are expressly encompassed bythe present invention.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and 0-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like. Biologically active molecules encompasses a variety ofpolypeptides including, but not limited to, Representative non-limitingclasses of polypeptides useful in the present invention include thosefalling into the following therapeutic categories: adrenocorticotropichormone peptides, adrenomedullin peptides, allatostatin peptides, amylinpeptides, amyloid beta-protein fragment peptides, angiotensin peptides,antibiotic peptides, antigenic polypeptides, anti-microbial peptides,apoptosis related peptides, atrial natriuretic peptides, bag cellpeptides, bombesin peptides, bone GLA peptides, bradykinin peptides,brain natriuretic peptides, C-peptides, C-type natriuretic peptides,calcitonin peptides, calcitonin gene related peptides, CART peptides,casomorphin peptides, chemotactic peptides, cholecystokinin peptides,colony-stimulating factor peptides, corticortropin releasing factorpeptides, cortistatin peptides, cytokine peptides, dermorphin peptides,dynorphin peptides, endorphin peptides, endothelin peptides, ETareceptor antagonist peptides, ETb receptor antagonist peptides,enkephalin peptides, fibronectin peptides, galanin peptides, gastrinpeptides, glucagon peptides, Gn-RH associated peptides, growth factorpeptides, growth hormone peptides, GTP-binding protein fragmentpeptides, guanylin peptides, inhibin peptides, insulin peptides,interleukin peptides, laminin peptides, leptin peptides, leucokininpeptides, luteinizing hormone-releasing hormone peptides, mastoparanpeptides, mast cell degranulating peptides, melanocyte stimulatinghormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides. (see U.S. Pat. No. 6,858,580).

Examples of biologically active molecules that are polypeptides include,but are not limited to, pituitary hormones such as vasopressin,oxytocin, melanocyte stimulating hormones, adrenocorticotropic hormones,growth hormones; hypothalamic hormones such as growth hormone releasingfactor, corticotropin releasing factor, prolactin releasing peptides,gonadotropin releasing hormone and its associated peptides, luteinizinghormone release hormones, thyrotropin releasing hormone, orexins, andsomatostatin; thyroid hormones such as calcitonins, calcitoninprecursors, and calcitonin gene related peptides; parathyroid hormonesand their related proteins; pancreatic hormones such as insulin andinsulin-like peptides, glucagon, somatostatin, pancreatic polypeptides,amylin, peptide YY, and neuropeptide Y; digestive hormones such asgastrin, gastrin releasing peptides, gastrin inhibitory peptides,cholecystokinin, secretin, motilin, and vasoactive intestinal peptide;natriuretic peptides such as atrial natriuretic peptides, brainnatriuretic peptides, and C-type natriuretic peptides; neurokinins suchas neurokinin A, neurokinin B, and substance P; renin related peptidessuch as renin substrates and inhibitors and angiotensins; endothelins,including big endothelin, endothelin A receptor antagonists, andsarafotoxin peptides; and other peptides such as adrenomedullinpeptides, allatostatin peptides, amyloid beta protein fragments,antibiotic and antimicrobial peptides, apoptosis related peptides, bagcell peptides, bombesin, bone Gla protein peptides, CART peptides,chemotactic peptides, cortistatin peptides, fibronectin fragments andfibrin related peptides, FMRF and analog peptides, galanin and relatedpeptides, growth factors and related peptides, G therapeuticpeptide-binding protein fragments, guanylin and uroguanylin, inhibinpeptides, interleukin and interleukin receptor proteins, lamininfragments, leptin fragment peptides, leucokinins, mast celldegranulating peptides, pituitary adenylate cyclase activatingpolypeptides, pancreastatin, peptide T, polypeptides, virus relatedpeptides, signal transduction reagents, toxins, and miscellaneouspeptides such as adjuvant peptide analogs, alpha mating factor,antiarrhythmic peptide, antifreeze polypeptide, anorexigenic peptide,bovine pineal antireproductive peptide, bursin, C3 peptide P16, tumornecrosis factor, cadherin peptide, chromogranin A fragment,contraceptive tetrapeptide, conantokin G, conantokin T, crustaceancardioactive peptide, C-telopeptide, cytochrome b588 peptide, decorsin,delicious peptide, delta-sleep-inducing peptide, diazempam-bindinginhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide,platelet calpain inhibitor (P1), plasminogen activator inhibitor 1,rigin, schizophrenia related peptide, serum thymic factor, sodiumpotassium A therapeutic peptidease inhibitor-1, speract, spermactivating peptide, systemin, thrombin receptor agonists, thymic humoralgamma2 factor, thymopentin, thymosin alpha 1, thymus factor, tuftsin,adipokinetic hormone, uremic pentapeptide, glucose-dependentinsulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1),glucagon-like peptide-2 (GLP-1), exendin-3, exendin-4, and othertherapeutic peptides or fragments thereof. Additional examples ofpeptides include ghrelin, opioid peptides (casomorphin peptides,demorphins, endorphins, enkephalins, deltorphins, dynorphins, andanalogs and derivatives of these), thymic peptides (thymopoietin,thymulin, thymopentin, thymosin, Thymic Humoral Factor (THF)), celladhesion peptides, complement inhibitors, thrombin inhibitors, trypsininhibitors, alpha-1 antitrypsin, Sea Urchin Sperm Activating Peptide,SHU-9119 MC3-R & MC4-R Antagonist, glaspimod (immunostimulant, usefulagainst bacterial infections, fungal infections, immune deficiencyimmune disorder, leukopenia), HP-228 (melanocortin, useful againstchemotherapy induced emesis, toxicity, pain, diabetes mellitus,inflammation, rheumatoid arthritis, obesity), alpha 2-plasmin inhibitor(plasmin inhibitor), APC tumor suppressor (tumor suppressor, usefulagainst neoplasm), early pregnancy factor (immunosuppressor), endozepinediazepam binding inhibitor (receptor peptide), gamma interferon (usefulagainst leukemia), glandular kallikrein-1 (immunostimulant), placentalribonuclease inhibitor, sarcolecin binding protein, surfactant proteinD, Wilms' tumor suppressor, GABAB lb receptor peptide, prion relatedpeptide (iPrP13), choline binding protein fragment (bacterial relatedpeptide), telomerase inhibitor, cardiostatin peptide, endostatin derivedpeptide (angiogenesis inhibitor), prion inhibiting peptide, N-methylD-aspartate receptor antagonist, C-peptide analog (useful againstdiabetic complications), RANTES, NTY receptors, NPY2-R (neuropeptide Ytype 2-receptor) ligands, NC4R peptides, or fragments thereof. See U.S.Pat.No. 6,849,714 which is incorporated by reference herein. Alsoincluded are Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor,antibodies, Apolipoprotein, Apoprotein, Atrial natriuretic factor,Atrial natriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GRO□/MGSA, GRO, GRQ, MIP-1, MIP-1, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”), Exfoliating toxins A and B, Factor IX,Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF),Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase,Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian,Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hirudin, Humanserum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons(e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones(e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G,Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complementreceptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3,4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens,i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED,SEE), Superoxide dismutase, Toxic shock syndrome toxin (TSST-1),Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factorbeta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosisfactor-alpha (TNF alpha), Vascular Endothelial Growth Factor (VEGEF),Urokinase, T-20, SS-14, LHRH, erythropoietin (EPO), G-CSF, TPO, axokine,leptin, and many others. Examples of hA conjugated, linked, or fused tobiologically active molecules may be found in U.S. Pat. Nos. 7,056,701;7,041,478; 7,045,318; 6,994,857; 6,987,006; 6,972,322; 6,946,134;6,926,898; 6,905,688; 6,686,179; 6,548,653; 6,423,512; 5,773,417; and5,594,110, which are incorporated by reference herein.

A “bifunctional polymer” or “bifunctional linker” refers to a moleculecomprising two discrete functional groups that are capable of reactingspecifically with other moieties (including but not limited to, aminoacid side groups) to form covalent or non-covalent linkages. Abifunctional linker having one functional group reactive with a group ona particular biologically active component, and another group reactivewith a group on a second biological component, may be used to form aconjugate that includes the first biologically active component, thebifunctional linker and the second biologically active component. Manyprocedures and linker molecules for attachment of various compounds topeptides are known. See, e.g., European Patent Application No. 188,256;U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338;and 4,569,789 which are incorporated by reference herein. A“multi-functional polymer” or “mulit-functional linker” refers to amolecule comprising two or more discrete functional groups that arecapable of reacting specifically with other moieties (including but notlimited to, amino acid side groups) to form covalent or non-covalentlinkages. A bi-functional polymer or linker, or a multi-functionalpolymer or linker may be any desired length or molecular weight, and maybe selected to provide a particular desired spacing or conformationbetween one or more molecules linked to the hPP or hFc and its bindingpartner or the hPP or hFc.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C ₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3, —C(O)NR2, —(C₁-C₁₀aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl),—(CH₂)_(m)—O—(—(CH₂)_(m)—O(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O) NR₂, —NRC(S)NR₂, salts thereof, andthe like. Each R as used herein is H, alkyl or substituted alkyl, arylor substituted aryl, aralkyl, or alkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′-represents both —C(O)₂R′- and—R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto hPP or hA or hFc polypeptides can result in changes including, butnot limited to, increased or modulated serum half-life, or increased ormodulated therapeutic half-life relative to the unmodified form,modulated immunogenicity, modulated physical association characteristicssuch as aggregation and multimer formation, altered receptor binding,altered binding to one or more binding partners, and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity, and may be utilized as a linkerfor attaching hPP or hA or hFc to other substances, including but notlimited to one or more hPP or hA or hFc polypeptides, or one or morebiologically active molecules. Suitable polymers include, but are notlimited to, polyethylene glycol, polyethylene glycol propionaldehyde,mono Cl-C10 alkoxy or aryloxy derivatives thereof (described in U.S.Pat. No. 5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (po ly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to, from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified hPP or hA orhFc relative to its non-modified form. Serum half-life is measured bytaking blood samples at various time points after administration of hPPor hA or hFc, and determining the concentration of that molecule in eachsample. Correlation of the serum concentration with time allowscalculation of the serum half-life. Increased serum half-life desirablyhas at least about two-fold, but a smaller increase may be useful, forexample where it enables a satisfactory dosing regimen or avoids a toxiceffect. In some embodiments, the increase is at least about three-fold,at least about five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of hPP or hA or hFc, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins: Structures and Molecular Properties    (W H Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment.

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³H, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION I. Introduction

The hPP or hFc molecules comprising at least one unnatural amino acidare provided in the present invention. In certain embodiments of theinvention, the hPP or hFc polypeptide with at least one unnatural aminoacid includes at least one post-translational modification. In someembodiments the hPP is hA. In one embodiment, the at least onepost-translational modification of the hPP or hA or hFc comprisesattachment of a molecule including but not limited to, a label, a dye, apolymer, a water-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2] cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified hPP or hA or hFcpolypeptide of the present invention, at least one unnatural amino acid(including but not limited to, unnatural amino acid containing a ketofunctional group) comprising at least one post-translationalmodification, is used where the at least one post-translationalmodification comprises a saccharide moiety. In certain embodiments, thepost-translational modification is made in vivo in a eukaryotic cell orin a non-eukaryotic cell. A linker, polymer, water soluble polymer, orother molecule may attach the molecule to the polypeptide. The moleculemay be linked directly to the polypeptide.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on membersof the hPP family, in particular hA, or hFc comprising at least onenon-naturally encoded amino acid. Introduction of at least onenon-naturally encoded amino acid into an hPP or hA or hFc family membercan allow for the application of conjugation chemistries that involvespecific chemical reactions, including, but not limited to, with one ormore non-naturally encoded amino acids while not reacting with thecommonly occurring 20 amino acids. In some embodiments, the hPP or hA orhFc family member comprising the non-naturally encoded amino acid islinked to a water soluble polymer, such as polyethylene glycol (PEG),via the side chain of the non-naturally encoded amino acid. Thisinvention provides a highly efficient method for the selectivemodification of proteins by coupling the protein with other moleculesincluding but not limited to polymers, linkers, or biologically activemolecules, which involves the selective incorporation of non-geneticallyencoded amino acids, including but not limited to, those amino acidscontaining functional groups or substituents not found in the 20naturally incorporated amino acids, including but not limited to aketone, an azide or acetylene moiety, into proteins in response to aselector codon and the subsequent modification of those amino acids witha suitably reactive molecule. Once incorporated, the amino acid sidechains can then be modified by utilizing chemistry methodologies knownto those of ordinary skill in the art to be suitable for the particularfunctional groups or substituents present in the non-naturally encodedamino acid. Known chemistry methodologies of a wide variety are suitablefor use in the present invention to couple molecules to the protein.Such methodologies include but are not limited to a Huisgen [3+2]cycloaddition reaction (see, e.g., Padwa, A. in Comprehensive OrganicSynthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but notlimited to, acetylene or azide derivatives, respectively.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2] cycloaddition product.

II. Human Plasma Protein Family

As used herein, “human plasma protein or polypeptide” or “hPP” includesthose polypeptides and proteins that are found in normal human bloodplasma, including hPP analogs, hPP isoforms, hPP mimetics, hPPfragments, hybrid hPP proteins, fusion proteins, oligomers andmultimers, homologues, glycosylation pattern variants, variants, splicevariants, and muteins, thereof, regardless of the biological activity ofsame, and further regardless of the method of synthesis or manufacturethereof including, but not limited to, recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), invitro, in vivo, by microinjection of nucleic acid molecules, synthetic,transgenic, and gene activated methods. A variety of hPP's are known inthe art and can be found in Anderson et al., Molecular & CellularProteomics, 3.4:311-326 (2004); and Ping et al, Proteomics, 5:3506-3519(2005), which are incorporated by reference herein.

Additional members of the hPP family are likely to be discovered in thefuture. New members of the hPP famil y can be identified throughcomputer-aided secondary and tertiary structure analyses of thepredicted protein sequences, and by selection techniques designed toidentify molecules that bind to a particular target. Members of the hPPsupergene family typically possess four or five amphipathic helicesjoined by non-helical amino acids (the loop regions). The proteins maycontain a hydrophobic signal sequence at their N-terminus to promotesecretion from the cell. Such later discovered members of the hPPsupergene family also are included within this invention.

Thus, the description of the hPP family or hA is provided forillustrative purposes and by way of example only and not as a limit onthe scope of the methods, compositions, strategies and techniquesdescribed herein. Further, reference to hPP or hA polypeptides in thisapplication is intended to use the generic term as an example of anymember of the hPP family. Thus, it is understood that the modificationsand chemistries described herein with reference to hPP or hApolypeptides or protein can be equally applied to any member of the hPPfamily, including those specifically listed herein.

III. General Recombinant Nucleic Acid Methods For Use With The Invention

In numerous embodiments of the present invention, nucleic acids encodingan hPP polypeptide of interest will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom an hPP or hFc polypeptide. In some embodiments, the sequencesencoding the polypeptides of the invention are operably linked to aheterologous promoter. Isolation of hPP and production of hPP in hostcells are described in, e.g., U.S. Pat. Nos. 5,648,243; 5,707,828 and5,521,287, which are incorporated by reference herein.

A nucleotide sequence encoding an hPP polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 1 and thenchanging the nucleotide sequence so as to effect introduction (i.e.,incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). A nucleotidesequence encoding an hFc polypeptide comprising a non-naturally encodedamino acid may be synthesized on the basis of the amino acid sequence ofthe parent polypeptide, including but not limited to, having the aminoacid sequence shown in SEQ ID NO: 22 and then changing the nucleotidesequence so as to effect introduction (i.e., incorporation orsubstitution) or removal (i.e., deletion or substitution) of therelevant amino acid residue(s). The nucleotide sequence may beconveniently modified by site-directed mutagenesis in accordance withconventional methods. Alternatively, the nucleotide sequence may beprepared by chemical synthesis, including but not limited to, by usingan oligonucleotide synthesizer, wherein oligonucleotides are designedbased on the amino acid sequence of the desired polypeptide, andpreferably selecting those codons that are favored in the host cell inwhich the recombinant polypeptide will be produced. For example, severalsmall oligonucleotides coding for portions of the desired polypeptidemay be synthesized and assembled by PCR, ligation or ligation chainreaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88: 189-193(1991); U.S. Pat. No. 6,521,427 which are incorporated by referenceherein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, or any combination thereof. Additional suitablemethods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Arm. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′Exonu cleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundstromet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley- Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the hPP polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O—RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988),5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res 16:791-802. When the O—RS, O-tRNA and thenucleic acid that encodes the polypeptide of interest are combined invivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods known to one of ordinary skill in the art and describedherein to include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as an hPPpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into an hPP polypeptide. In general, theintroduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, an hPP polypeptide that includes a non-naturally encoded aminoacid containing an azido functional group can be reacted with a polymer(including but not limited to, poly(ethylene glycol) or, alternatively,a second polypeptide containing an alkyne moiety to form a stableconjugate resulting for the selective reaction of the azide and thealkyne functional groups to form a Huisgen [3+2] cycloaddition product.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occuring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occuring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Patent Application Publications 2003/0082575 and2003/0108885, which are incorporated by reference herein. In addition tounnatural amino acids that contain novel side chains, unnatural aminoacids that may be suitable for use in the present invention alsooptionally comprise modified backbone structures, including but notlimited to, as illustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-0-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4 ,6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and y-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆- C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, y-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L- phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAciβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs.

In one embodiment, compositions of an hPP polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3′OH or a 2′OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2″^(d) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Structure and Synthesis of Non-Natural Amino Acids: CarbonylCarbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present invention provides hPP or hA or hFclinked to a water soluble polymer, e.g., a PEG, by an oxime bond.

Many types of non-naturally encoded amino acids are suitable forformation of oxime bonds. These include, but are not limited to,non-naturally encoded amino acids containing a carbonyl, dicarbonyl, orhydroxylamine group. Such amino acids are described in U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” which areincorporated herein by reference in their entirety. Non-naturallyencoded amino acids are also described in U.S. Pat. No. 7,083,970 andU.S. Pat. No. 7,045,337, which are incorporated by reference herein intheir entirety.

Some embodiments of the invention utilize hPP or hA or hFc polypeptidesthat are substituted at one or more positions with apara-acetylphenylalanine amino acid. The synthesis ofp-acetyl-(+/−)-phenylalanine and m-acetyl-(+/−)-phenylalanine aredescribed in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003),incorporated by reference. Other carbonyl- or dicarbonyl-containingamino acids can be similarly prepared by one of ordinary skill in theart. Further, non-limiting examplary syntheses of non-natural amino acidthat are included herein are presented in FIGS. 4, 24-34 and 36-39 ofU.S. Pat. No. 7,083,970, which is incorporated by reference herein inits entirety.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a carbonyl group(including a keto group and a dicarbonyl group), a carbonyl-like group(which has reactivity similar to a carbonyl group (including a ketogroup and a dicarbonyl group) and is structurally similar to a carbonylgroup), a masked carbonyl group (which can be readily converted into acarbonyl group (including a keto group and a dicarbonyl group)), or aprotected carbonyl group (which has reactivity similar to a carbonylgroup (including a keto group and a dicarbonyl group) upondeprotection). Such amino acids include amino acids having the structureof Formula (IV):

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, 13 CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl;

-   J is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   each R″ is independently H, alkyl, substituted alkyl, or a    protecting group, or when more than one R″ group is present, two R″    optionally form a heterocycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   or the -A-B-J-R groups together form a bicyclic or tricyclic    cycloalkyl or heterocycloalkyl comprising at least one carbonyl    group, including a dicarbonyl group, protected carbonyl group,    including a protected dicarbonyl group, or masked carbonyl group,    including a masked dicarbonyl group;-   or the -J-R group together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group;-   with a proviso that when A is phenylene and each R₃ is H, B is    present; and that when A is —(CH₂)₄— and each R₃ is H, B is not    —NHC(O)(CH₂CH₂)—; and that when A and B are absent and each R₃ is H,    R is not methyl.

In addition, having the structure of Formula (V) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   with a proviso that when A is phenylene, B is present; and that when    A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B are    absent, R is not methyl.

In addition, amino acids having the structure of Formula (VI) areincluded:

wherein:

-   B is a linker selected from the group consisting of lower alkylene,    substituted lower alkylene, lower alkenylene, substituted lower    alkenylene, lower heteroalkylene, substituted lower heteroalkylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,    —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene    or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted    alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted    alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,    —N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)C(O)O—,    —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR', and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected group, carboxylprotected or a salt thereof. In addition, any of the followingnon-natural amino acids may be incorporated into a non-natural aminoacid polypeptide.

In addition, the following amino acids having the structure of Formula(VII) are included:

wherein

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8;-   with a proviso that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N)R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(IX) are included:

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   wherein each R_(a) is independently selected from the group    consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,    —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XI) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(XII) are included:

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   wherein each R_(a) is independently selected from the group    consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,    —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIII) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIV) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynuclebtide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and    R⁹ on each CR⁸R⁹ group independently selected from the group    consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸    and R⁹ can together form ═O or a cycloalkyl, or any to adjacent R⁸    groups can together form a cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group is    independently selected from the group consisting of H, alkoxy,    alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form    ═O or a cycloalkyl, or any to adjacent R⁸ groups can together form a    cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group is    independently selected from the group consisting of H, alkoxy,    alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form    ═O or a cycloalkyl, or any to adjacent R⁸ groups can together form a    cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-A) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-B) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (XVII) areincluded:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   M is

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl;-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,    substituted alkyl, cycloalkyl, or substituted cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide.

In addition, amino acids having the structure of Formula (XVIII) areincluded:

wherein:

-   M is

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl;-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,    substituted alkyl, cycloalkyl, or substituted cycloalkyl;-   R₁ is optional, and when present, is H, an amino protecting group,    resin, amino acid, polypeptide, or polynucleotide; and-   R₂ is optional, and when present, is OH, an ester protecting group,    resin, amino acid, polypeptide, or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl.

In addition, amino acids having the structure of Formula (XIX) areincluded:

wherein:

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl; and-   T₃ is O, or S.

In addition, amino acids having the structure of Formula (XX) areincluded:

wherein:

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl.

In addition, the following amino acids having structures of Formula(XXI) are included:

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-natural amino acid bearing adjacenthydroxyl and amino groups can be incorporated into the polypeptide as a“masked” aldehyde functionality. For example, 5-hydroxylysine bears ahydroxyl group adjacent to the epsilon amine. Reaction conditions forgenerating the aldehyde typically involve addition of molar excess ofsodium metaperiodate under mild conditions to avoid oxidation at othersites within the polypeptide. The pH of the oxidation reaction istypically about 7.0. A typical reaction involves the addition of about1.5 molar excess of sodium meta periodate to a buffered solution of thepolypeptide, followed by incubation for about 10 minutes in the dark.See, e.g. U.S. Pat. No. 6,423,685.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl ordicarbonyl group allows for selective modification in the presence ofthe other amino acid side chains. See, e.g., Cornish, V. W., et al., J.Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science276:1125-1128 (1997).

Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. Provisional Patent Application No. 60/638,418 is incorporated byreference in its entirety. Thus, the disclosures provided in Section V(entitled “Non-natural Amino Acids”), Part B (entitled “Structure andSynthesis of Non-Natural Amino Acids: Hydroxylamine-Containing AminoAcids”), in U.S. Provisional Patent Application No. 60/638,418 applyfully to the methods, compositions (including Formulas I-XXXV),techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein to thesame extent as if such disclosures were fully presented herein. U.S.Patent Publication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 andWO 2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” are also incorporatedherein by reference in their entirety.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL-and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide -containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-y-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containinghPPpolypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the hPP polypeptide comprises anon-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated intohPPpolypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to an hPP polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

F. Additional Reactive Groups

Additional reactive groups and non-naturally encoded amino acids,including but not limited to para-amino-phenylalanine, that can beincorporated into hPP or hA or hFc of the invention are described in thefollowing patent applications which are all incorporated by reference intheir entirety herein: U.S. Patent Publication No. 2006/0194256, U.S.Patent Publication No. 2006/0217532, U.S. Patent Publication No.2006/0217289, U.S. Provisional Patent No. 60/755,338; U.S. ProvisionalPatent No. 60/755,711; U.S. Provisional Patent No. 60/755,018;International Patent Application No. PCT/US06/49397; WO 2006/069246;U.S. Provisional Patent No. 60/743,041; U.S. Provisional Patent No.60/743,040; International Patent Application No. PCT/US06/47822; U.S.Provisional Patent No. 60/882,819; U.S. Provisional Patent No.60/882,500; and U.S. Provisional Patent No. 60/870,594. Theseapplications also discuss reactive groups that may be present on PEG orother polymers, including but not limited to, hydroxylamine (aminooxy)groups for conjugation.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 1 EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type BaseStructure High-mannose

Hybrid

Complex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like. U.S. Pat. Nos.4,963,495 and 6,436,674, which are incorporated herein by reference,detail constructs that may improve secretion of hPP polypeptides.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In vivo Generation of hPP or hA or hFc ComprisingNon-Genetically-Encoded Amino Acids

The hPP or hFc polypeptides of the invention can be generated in vivousing modified tRNA and tRNA synthetases to add to or substitute aminoacids that are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. No. 7,045,337 and U.S. Patent Application Publication2003/0108885 (Ser. No. 10/126,931) which are incorporated by referenceherein. These methods involve generating a translational machinery thatfunctions independently of the synthetases and tRNAs endogenous to thetranslation system (and are therefore sometimes referred to as“orthogonal”). Typically, the translation system comprises an orthogonaltRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS).Typically, the O-RS preferentially aminoacylates the O-tRNA with atleast one non-naturally occurring amino acid in the translation systemand the O-tRNA recognizes at least one selector codon that is notrecognized by other tRNAs in the system. The translation system thusinserts the non-naturally-encoded amino acid into a protein produced inthe system, in response to an encoded selector codon, thereby“substituting” an amino acid into a position in the encoded polypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O-RSs arealso described in U.S. Pat. No. 7,045,337 and U.S. Patent ApplicationPublication 2003/0108885 (Serial No. 10/126,931) which are incorporatedby reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O-RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. Pat. No.7,045,337 which is incorporated by reference herein. O-RS and O-tRNAthat incorporate both keto- and azide-containing amino acids in S.cerevisiae are described in Chin, J. W., et al., Science 301:964-967(2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A.98:2268-2273) and tyrosyl (see,e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the hPP polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. Pat. No.7,045,337, which is incorporated by reference herein. Methods forselecting an orthogonal tRNA-tRNA synthetase pair for use in in vivotranslation system of an organism are also described in U.S. Pat. No.7,045,337 and U.S. Patent Application Publication 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein. PCT PublicationNo. WO 04/035743 entitled “Site Specific Incorporation of Keto AminoAcids into Proteins,” which is incorporated by reference herein in itsentirety, describes orthogonal RS and tRNA pairs for the incorporationof keto amino acids. PCT Publication No. WO 04/094593 entitled“Expanding the Eukaryotic Genetic Code,” which is incorporated byreference herein in its entirety, describes orthogonal RS and tRNA pairsfor the incorporation of non-naturally encoded amino acids in eukaryotichost cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O-RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O-RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O-RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O-RS, wherein the at least one recombinant O-RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O-RS derived from at least one recombinant O-RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O-RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O-RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O-RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O-RS pair, wherein the at least onespecific O-tRNA/O-RS pair comprises at least one recombinant O-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O-RS pairs produced by themethods are included. For example, the specific O-tRNA/O-RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in hPP or hA or hFc

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into hPP or hFc polypeptides. One ormore non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the hPP or hFc polypeptide. It is readily apparent tothose of ordinary skill in the art that any position of the polypeptidechain is suitable for selection to incorporate a non-naturally encodedamino acid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing an hPP or hFc molecule having any desiredproperty or activity, including but not limited to, agonists,super-agonists, inverse agonists, antagonists, receptor bindingmodulators, receptor activity modulators, modulators of binding tobinding partners, binder partner activity modulators, binding partnerconformation modulators, dimer or multimer formation, no change toactivity or property compared to the native molecule, or manipulatingany physical or chemical property of the polypeptide such as solubility,aggregation, or stability. For example, locations in the polypeptiderequired for biological activity of hPP or hFc polypeptides can beidentified using point mutation analysis, alanine scanning or homologscanning methods known in the art. U.S. Pat. Nos. 5,580,723; 5,834,250;6,013,478; 6,428,954; and 6,451,561, which are incorporated by referenceherein, describe methods for the systematic analysis of the structureand function of polypeptides such as hGH by identifying active domainswhich influence the activity of the polypeptide with a target substance.Residues other than those identified as critical to biological activityby alanine or homolog scanning mutagenesis may be good candidates forsubstitution with a non-naturally encoded amino acid depending on thedesired activity sought for the polypeptide. Alternatively, the sitesidentified as critical to biological activity may also be goodcandidates for substitution with a non-naturally encoded amino acid,again depending on the desired activity sought for the polypeptide.Another alternative would be to simply make serial substitutions in eachposition on the polypeptide chain with a non-naturally encoded aminoacid and observe the effect on the activities of the polypeptide. It isreadily apparent to those of ordinary skill in the art that any means,technique, or method for selecting a position for substitution with anon-natural amino acid into any polypeptide is suitable for use in thepresent invention.

The structure and activity of naturally-occurring mutants of hPPpolypeptides that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-naturally encoded amino acid. Once residues that are likelyto be intolerant to substitution with non-naturally encoded amino acidshave been eliminated, the impact of proposed substitutions at each ofthe remaining positions can be examined from the three-dimensionalcrystal structure of the hPP or hFc and its binding proteins. Models maybe made investigating the secondary and tertiary structure ofpolypeptides, if three-dimensional structural data is not available.Thus, those of ordinary skill in the art can readily identify amino acidpositions that can be substituted with non-naturally encoded aminoacids.

In some embodiments, the hPP or hFc polypeptides of the inventioncomprise one or more non-naturally occurring amino acids positioned in aregion of the protein that does not disrupt the secondary structure ofthe polypeptide.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in hA as follows: before position1 (i.e. at the N-terminus), 17, 34, 55, 56, 58, 60, 81, 82, 86, 92, 94,111, 114, 116, 119, 129, 170, 172, 173, 276, 277, 280, 297, 300, 301,313, 317, 321, 362, 363, 364, 365, 368, 375, 397, 439, 442, 495, 496,498, 500, 501, 505, 515, 538, 541, 542, 560, 562, 564, 574, 581, andafter position 582 (i.e., at the carboxyl terminus of the protein),.(SEQ ID NO: 1).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in an hPP or hFcpolypeptide. In general, a particular non-naturally encoded amino acidis selected for incorporation based on an examination of the threedimensional crystal structure of an hPP or hFc polypeptide with itsreceptor or binding partner, a preference for conservative substitutions(i.e., aryl-based non-naturally encoded amino acids, such asp-acetylphenylalanine or O-propargyltyrosine substituting for Phe, Tyror Trp), and the specific conjugation chemistry that one desires tointroduce into the hPP or hFc polypeptide (e.g., the introduction of4-azidophenylalanine if one wants to effect a Huisgen [3+2]cycloaddition with a water soluble polymer bearing an alkyne moiety or aamide bond formation with a water soluble polymer that bears an arylester that, in turn, incorporates a phosphine moiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2] cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within thehPP polypeptide to affect other biological traits of the hPP or hFcpolypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the hPP or hFc polypeptide orincrease affinity of the hPP or hFc polypeptide for its receptor orbinding partner. In some cases, the other additions, substitutions ordeletions may increase the solubility (including but not limited to,when expressed in E. coli or other host cells) of the hPP or hFcpolypeptide. In some embodiments additions, substitutions or deletionsmay increase the polypeptide solubility following expression in E. colior other recombinant host cells. In some embodiments sites are selectedfor substitution with a naturally encoded or non-natural amino acid inaddition to another site for incorporation of a non-natural amino acidthat results in increasing the polypeptide solubility followingexpression in E. coli or other recombinant host cells. In someembodiments, the hPP or hFc polypeptides comprise another addition,substitution or deletion that modulates affinity for the hPP or hFcpolypeptide receptor, binding proteins, or associated ligand, modulates(including but not limited to, increases or decreases) receptordimerization, stabilizes receptor dimers, modulates circulatinghalf-life, modulates release or bio-availability, facilitatespurification, or improves or alters a particular route ofadministration. Similarly, hPP or hFc polypeptides can comprise chemicalor enzyme cleavage sequences, protease cleavage sequences, reactivegroups, antibody-binding domains (including but not limited to, FLAG orpoly-His) or other affinity based sequences (including, but not limitedto, FLAG, poly-His, GST, etc.) or linked molecules (including, but notlimited to, biotin) that improve detection (including, but not limitedto, GFP), purification, transport through tissues or cell membranes,prodrug release or activation, hPP or hFc size reduction, or othertraits of the polypeptide.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the hPP or hFc polypeptide further includes 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more substitutions of one or more non-naturally encodedamino acids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in the following regions of hA aresubstituted with one or more non-naturally encoded amino acids: beforeposition 1 (i.e. at the N-terminus), 17, 34, 55, 56, 58, 60, 81, 82, 86,92, 94, 111, 114, 116, 119, 129, 170, 172, 173, 276, 277, 280, 297, 300,301, 313, 317, 321, 362, 363, 364, 365, 368, 375, 397, 439, 442, 495,496, 498, 500, 501, 505, 515, 538, 541, 542, 560, 562, 564, 574, 581,and after position 582 (i.e., at the carboxyl terminus of the protein),.(SEQ ID NO: 1).

In some cases, the one or more non-naturally encoded residues are linkedto one or more lower molecular weight linear or branched PEGs(approximately ˜5-20 kDa in mass or less), thereby enhancing bindingaffinity and comparable serum half-life relative to the species attachedto a single, higher molecular weight PEG.

Preferred sites for incorporation in hA of two or more non-naturallyencoded amino acids include combinations of the following residues:before position 1 (i.e. at the N-terminus), 17, 34, 55, 56, 58, 60, 81,82, 86, 92, 94, 111, 114, 116, 119, 129, 170, 172, 173, 276, 277, 280,297, 300, 301, 313, 317, 321, 362, 363, 364, 365, 368, 375, 397, 439,442, 495, 496, 498, 500, 501, 505, 515, 538, 541, 542, 560, 562, 564,574, 581, and after position 582 (i.e., at the carboxyl terminus of theprotein),. (SEQ ID NO: 1).

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned hPP or hA or hFcpolynucleotide, one typically subclones polynucleotides encoding an hPPor hA or hFc polypeptide of the invention into an expression vector thatcontains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are known to those of ordinary skill in theart and described, e.g., in Sambrook et al. and Ausubel et al.

Bacterial expression systems for expressing hPP or hA or hFcpolypeptides of the invention are available in, including but notlimited to, E. coli, Bacillus sp., Pseudomonas fluorescens, Pseudomonasaeruginosa, Pseudomonas putida, and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells areknown to those of ordinary skill in the art and are also commerciallyavailable. In cases where orthogonal tRNAs and aminoacyl tRNAsynthetases (described above) are used to express the hPP or hApolypeptides of the invention, host cells for expression are selectedbased on their ability to use the orthogonal components. Exemplary hostcells include Gram-positive bacteria (including but not limited to B.brevis, B. subtilis, or Streptomyces) and Gram-negative bacteria (E.coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonasputida), as well as yeast and other eukaryotic cells. Cells comprisingO-tRNA/O-RS pairs can be used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 n1 to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

A number of vectors suitable for expression of hPP or hFc arecommercially available. Useful expression vectors for eukaryotic hosts,include but are not limited to, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Such vectors include pCDNA3.1(+)\Hyg (Invitrogen,Carlsbad, Calif., USA) and pCI-neo (Stratagene, La Jolla, Calif., USA).Bacterial plasmids, such as plasmids from E. coli, including pBR322,pET3a and pET12a, wider host range plasmids, such as RP4, phage DNAs,e.g., the numerous derivatives of phage lambda, e.g., NM989, and otherDNA phages, such as M13 and filamentous single stranded DNA phages maybe used. The 2μ plasmid and derivatives thereof, the POT1 vector (U.S.Pat. No. 4,931,373 which is incorporated by reference), the pJSO37vector described in (Okkels, Ann. New York Aced. Sci. 782, 202 207,1996) and pPICZ A, B or C (Invitrogen) may be used with yeast hostcells. For insect cells, the vectors include but are not limited to,pVL941, pBG311 (Cate et al., “Isolation of the Bovine and Human Genesfor Mullerian Inhibiting Substance And Expression of the Human Gene InAnimal Cells”, Cell, 45, pp. 685 98 (1986), pBluebac 4.5 and pMelbac(Invitrogen, Carlsbad, Calif.).

The nucleotide sequence encoding a hPP or hFc polypeptide may or may notalso include sequence that encodes a signal peptide. The signal peptideis present when the polypeptide is to be secreted from the cells inwhich it is expressed. Such signal peptide may be any sequence. Thesignal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) J.Imm. Methods 152:89 104) describe a signal peptide for use in mammaliancells (murine 1 g kappa light chain signal peptide). Other signalpeptides include but are not limited to, the α-factor signal peptidefrom S. cerevisiae (U.S. Pat. No. 4,870,008 which is incorporated byreference herein), the signal peptide of mouse salivary amylase (O.Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modifiedcarboxypeptidase signal peptide (L. A. Valls et al., Cell 48, 1987, pp.887-897), the yeast BAR1 signal peptide (WO 87/02670, which isincorporated by reference herein), and the yeast aspartic protease 3(YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137).

Examples of suitable mammalian host cells are known to those of ordinaryskill in the art. Such host cells may be Chinese hamster ovary (CHO)cells, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cells (COS) (e.g. COS 1(ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), BabyHamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), andhuman cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells intissue culture. These cell lines and others are available from publicdepositories such as the American Type Culture Collection, Rockville,Md. In order to provide improved glycosylation of a hPP polypeptide, amammalian host cell may be modified to express sialyltransferase, e.g.1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335,which is incorporated by reference herein.

Methods for the introduction of exogenous DNA into mammalian host cellsinclude but are not limited to, calcium phosphate-mediated transfection,electroporation, DEAE-dextran mediated transfection, liposome-mediatedtransfection, viral vectors and the transfection methods described byLife Technologies Ltd, Paisley, UK using Lipofectamine 2000 and RocheDiagnostics Corporation, Indianapolis, USA using FuGENE 6. These methodsare well known in the art and are described by Ausbel et al. (eds.),1996, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, USA. The cultivation of mammalian cells may be performed accordingto established methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc.Totowa, N.J., USA and Harrison Mass. and Rae I F, General Techniques ofCell Culture, Cambridge University Press 1997).

I. Expression Systems, Culture, and Isolation

hPP or hA or hFc polypeptides may be expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding an hPP or hA or hFcpolypeptide. Such yeasts include, but are not limited to,ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts andyeasts belonging to the Fungi imperfecti (Blastomycetes) group. Theascosporogenous yeasts are divided into two families, Spermophthoraceaeand Saccharomycetaceae. The latter is comprised of four subfamilies,Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia,Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts includethe genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium,and Filobasidiella. Yeasts belonging to the Fungi Imperfecti(Blastomycetes) group are divided into two families, Sporobolomycetaceae(e.g., genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g.,genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of hPP or hA or hFcpolypeptides is within the skill of one of ordinary skill in the art. Inselecting yeast hosts for expression, suitable hosts may include thoseshown to have, for example, good secretion capacity, low proteolyticactivity, good secretion capacity, good soluble protein production, andoverall robustness. Yeast are generally available from a variety ofsources including, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding an hPP or hA polypeptide, areincluded in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. A CAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Pat. Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding an hPP or hA or hFcpolypeptide, are included in the progeny intended by this definition.

The selection of suitable insect cells for expression of hPP or hA orhFc polypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interalia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN No. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5N5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN No. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR B(1995); Kitts, NAR (1990) 18(19):5667;and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN No. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alfa, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are known to those of ordinary skill in the art. A widevariety of vectors are available for use in bacterial hosts. The vectorsmay be single copy or low or high multicopy vectors. Vectors may servefor cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., Nuc. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce hPP or hA polypeptides at high levels. Examples ofsuch vectors are known to those of ordinary skill in the art and includethe pET29 series from Novagen, and the pPOP vectors described inWO99/05297, which is incorporated by reference herein. Such expressionsystems produce high levels of hPP or hA or hFc polypeptides in the hostwithout compromising host cell viability or growth parameters. pET19(Novagen) is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. B(1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding an hPP or hA polypeptide, are included inthe progeny intended by this definition.

The selection of suitable host bacteria for expression of hPP or hApolypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com).

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of hPP or hA or hFc polypeptides. As will be apparent toone of skill in the art, the method of culture of the recombinant hostcell strain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are known to those of ordinaryskill in the art. Recombinant host cells are typically cultured inliquid medium containing assimilatable sources of carbon, nitrogen, andinorganic salts and, optionally, containing vitamins, amino acids,growth factors, and other proteinaceous culture supplements known tothose of ordinary skill in the art. Liquid media for culture of hostcells may optionally contain antibiotics or anti-fungals to prevent thegrowth of undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the hPP or hA or hFcpolypeptide accumulates intracellularly) or harvesting of culturesupernatant in either batch or continuous formats. For production inprokaryotic host cells, batch culture and cell harvest are preferred.

The hPP or hA or hFc polypeptides of the present invention are normallypurified after expression in recombinant systems. The hPP or hA or hFcpolypeptide may be purified from host cells or culture medium by avariety of methods known to the art. hPP or hA or hFc polypeptidesproduced in bacterial host cells may be poorly soluble or insoluble (inthe form of inclusion bodies). In one embodiment of the presentinvention, amino acid substitutions may readily be made in the hPP or hAor hFc polypeptide that are selected for the purpose of increasing thesolubility of the recombinantly produced protein utilizing the methodsdisclosed herein as well as those known in the art. In the case ofinsoluble protein, the protein may be collected from host cell lysatesby centrifugation and may further be followed by homogenization of thecells. In the case of poorly soluble protein, compounds including, butnot limited to, polyethylene imine (PEI) may be added to induce theprecipitation of partially soluble protein. The precipitated protein maythen be conveniently collected by centrifugation. Recombinant host cellsmay be disrupted or homogenized to release the inclusion bodies fromwithin the cells using a variety of methods known to those of ordinaryskill in the art. Host cell disruption or homogenization may beperformed using well known techniques including, but not limited to,enzymatic cell disruption, sonication, dounce homogenization, or highpressure release disruption. In one embodiment of the method of thepresent invention, the high pressure release technique is used todisrupt the E. coli host cells to release the inclusion bodies of thehPP or hA or hFc polypeptides. When handling inclusion bodies of hPP orhA or hFc polypeptide, it may be advantageous to minimize thehomogenization time on repetitions in order to maximize the yield ofinclusion bodies without loss due to factors such as solubilization,mechanical shearing or proteolysis.

Insoluble or precipitated hPP or hA or hFc polypeptide may then besolubilized using any of a number of suitable solubilization agentsknown to the art. The hPP or hA or hFc polyeptide may be solubilizedwith urea or guanidine hydrochloride. The volume of the solubilized hPPor hA or hFc polypeptide should be minimized so that large batches maybe produced using conveniently manageable batch sizes. This factor maybe significant in a large-scale commercial setting where the recombinanthost may be grown in batches that are thousands of liters in volume. Inaddition, when manufacturing hPP or hA or hFc polypeptide in alarge-scale commercial setting, in particular for human pharmaceuticaluses, the avoidance of harsh chemicals that can damage the machinery andcontainer, or the protein product itself, should be avoided, ifpossible. It has been shown in the method of the present invention thatthe milder denaturing agent urea can be used to solubilize the hPP or hAor hFc polypeptide inclusion bodies in place of the harsher denaturingagent guanidine hydrochloride. The use of urea significantly reduces therisk of damage to stainless steel equipment utilized in themanufacturing and purification process of hPP or hA or hFc polypeptidewhile efficiently solubilizing the hPP or hA or hFc polypeptideinclusion bodies.

In the case of soluble hPP or hA or hFc protein, the hPP or hA or hFcmay be secreted into the periplasmic space or into the culture medium.In addition, soluble hPP or hA or hFc may be present in the cytoplasm ofthe host cells. It may be desired to concentrate soluble hPP or hA orhFc prior to performing purification steps. Standard techniques known tothose of ordinary skill in the art may be used to concentrate solublehPP or hA or hFc from, for example, cell lysates or culture medium. Inaddition, standard techniques known to those of ordinary skill in theart may be used to disrupt host cells and release soluble hPP or hA orhFc from the cytoplasm or periplasmic space of the host cells.

When hPP or hA or hFc polypeptide is produced as a fusion protein, thefusion sequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved hPP or hA or hFc polypeptide may bepurified from the cleaved fusion sequence by methods known to those ofordinary skill in the art. Such methods will be determined by theidentity and properties of the fusion sequence and the hPP or hA or hFcpolypeptide, as will be apparent to one of ordinary skill in the art.Methods for purification may include, but are not limited to,size-exclusion chromatography, hydrophobic interaction chromatography,ion-exchange chromatography or dialysis or any combination thereof.

The hPP or hA or hFc polypeptide may also be purified to remove DNA fromthe protein solution. DNA may be removed by any suitable method known tothe art, such as precipitation or ion exchange chromatography, but maybe removed by precipitation with a nucleic acid precipitating agent,such as, but not limited to, protamine sulfate. The hPP or hA or hFcpolypeptide may be separated from the precipitated DNA using standardwell known methods including, but not limited to, centrifugation orfiltration. Removal of host nucleic acid molecules is an importantfactor in a setting where the hPP or hA or hFc polypeptide is to be usedto treat humans and the methods of the present invention reduce hostcell DNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human hPP or hA or hFc polypeptides of the invention can generally berecovered using methods standard in the art. For example, culture mediumor cell lysate can be centrifuged or filtered to remove cellular debris.The supernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the hPP or hA or hFcpolypeptide of the present invention includes separating deamidated andclipped forms of the hPP or hA or hFc polypeptide variant from theintact form.

Any of the following exemplary procedures can be employed forpurification of hPP or hA polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography;metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods known to those of ordinary skillin the art, including but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against unnatural aminoacids (or proteins or peptides comprising unnatural amino acids) areused as purification reagents, including but not limited to, foraffinity-based purification of proteins or peptides comprising one ormore unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein or polypeptide is optionally denatured and then renatured. Thisis accomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of hPP or hA or hFc polypeptide,the hPP or hA or hFc polypeptide thus produced may be misfolded and thuslacks or has reduced biological activity. The bioactivity of the proteinmay be restored by “refolding”. In general, misfolded hPP or hApolypeptide is refolded by solubilizing (where the hPP or hA or hFcpolypeptide is also insoluble), unfolding and reducing the polypeptidechain using, for example, one or more chaotropic agents (e.g. ureaand/or guanidine) and a reducing agent capable of reducing disulfidebonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At amoderate concentration of chaotrope, an oxidizing agent is then added(e.g., oxygen, cystine or cystamine), which allows the reformation ofdisulfide bonds. hPP or hA or hFc polypeptide may be refolded usingstandard methods known in the art, such as those described in U.S. Pat.Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated byreference herein. The hPP or hA or hFc polypeptide may also be cofoldedwith other proteins to form heterodimers or heteromultimers.

After refolding, the hPP or hA or hFc may be further purified.Purification of hPP or hA or hFc may be accomplished using a variety oftechniques known to those of ordinary skill in the art, includinghydrophobic interaction chromatography, size exclusion chromatography,ion exchange chromatography, reverse-phase high performance liquidchromatography, affinity chromatography, and the like or any combinationthereof. Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, hPP or hA or hFc may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, diafiltration and dialysis. hPP orhA or hFc that is provided as a single purified protein may be subjectto aggregation and precipitation.

The purified hPP or hA or hFc may be at least 90% pure (as measured byreverse phase high performance liquid chromatography, RP-HPLC, or sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or atleast 95% pure, or at least 98% pure, or at least 99% or greater pure.Regardless of the exact numerical value of the purity of the hPP or hAor hFc, the hPP or hA or hFc is sufficiently pure for use as apharmaceutical product or for further processing, such as conjugationwith a water soluble polymer such as PEG.

Certain hPP or hA or hFc molecules may be used as therapeutic agents inthe absence of other active ingredients or proteins (other thanexcipients, carriers, and stabilizers, serum albumin and the like), orthey may be complexed with another protein or a polymer.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate, extract, culture medium, inclusionbodies, periplasmic space of the host cells, cytoplasm of the hostcells, or other material, comprising hPP or hA or hFc polypeptide or onany hPP or hA or hFc polypeptide mixtures resulting from any isolationsteps including, but not limited to, affinity chromatography, ionexchange chromatography, hydrophobic interaction chromatography, gelfiltration chromatography, high performance liquid chromatography(“HPLC”), reversed phase-HPLC (“RP-HPLC”), expanded bed adsorption, orany combination and/or repetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the hPP or hAor hFc polypeptide may be reduced and denatured by first denaturing theresultant purified hPP or hA or hFc polypeptide in urea, followed bydilution into TRIS buffer containing a reducing agent (such as DTT) at asuitable pH. In another embodiment, the hPP or hA or hFc polypeptide isdenatured in urea in a concentration range of between about 2 M to about9 M, followed by dilution in TRIS buffer at a pH in the range of about5.0 to about 8.0. The refolding mixture of this embodiment may then beincubated. In one embodiment, the refolding mixture is incubated at roomtemperature for four to twenty-four hours. The reduced and denatured hPPor hA or hFc polypeptide mixture may then be further isolated orpurified.

As stated herein, the pH of the first hPP or hA or hFc polypeptidemixture may be adjusted prior to performing any subsequent isolationsteps. In addition, the first hPP or hA or hFc polypeptide mixture orany subsequent mixture thereof may be concentrated using techniquesknown in the art. Moreover, the elution buffer comprising the first hPPor hA or hFc polypeptide mixture or any subsequent mixture thereof maybe exchanged for a buffer suitable for the next isolation step usingtechniques known to those of ordinary skill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst hPP or hA or hFc polypeptide mixture. See generally ION EXCHANGECHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, AmershamBiosciences (Piscataway, N.J.)). Commercially available ion exchangecolumns include HITRAP®, HIPREP®, and HILOAD® Columns (AmershamBiosciences, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the hPP or hA or hFc polypeptide at any stage of thepurification process to isolate substantially purified hPP or hA or hFcpolypeptide. The cation exchange chromatography step may be performedusing any suitable cation exchange matrix. Useful cation exchangematrices include, but are not limited to, fibrous, porous, non-porous,microgranular, beaded, or cross-linked cation exchange matrix materials.Such cation exchange matrix materials include, but are not limited to,cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene,silica, polyether, or composites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding hPP or hA or hFc over a wide pH range. Weak cation exchangers,however, may lose ionization as a function of pH. For example, a weakcation exchanger may lose charge when the pH drops below about pH 4 orpH 5. Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having an hPP or hA or hFc binding pH rangeof about 2.5 to about 6.0. Alternatively, the strong cation exchangermay have an hPP or hA or hFc binding pH range of about pH 2.5 to aboutpH 5.5. The cation exchange matrix may be a strong cation exchangerhaving an hPP or hA or hFc binding pH of about 3.0. Alternatively, thecation exchange matrix may be a strong cation exchanger, preferablyhaving an hPP or hA or hFc binding pH range of about 6.0 to about 8.0.The cation exchange matrix may be a strong cation exchanger preferablyhaving an hPP or hA or hFc binding pH range of about 8.0 to about 12.5.Alternatively, the strong cation exchanger may have an hPP or hA bindingpH range of about pH 8.0 to about pH 12.0.

Prior to loading the hPP or hA or hFc, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the hPP or hA or hFc may be added and thecolumn may be washed one to several times, prior to elution ofsubstantially purified hPP or hA or hFc, also using a weak acid solutionsuch as a weak acetic acid or phosphoric acid solution. For example,approximately 2-4 column volumes of 20 mM acetic acid, pH 3, may be usedto wash the column. Additional washes using, e.g., 2-4 column volumes of0.05 M sodium acetate, pH 5.5, or 0.05 M sodium acetate mixed with 0.1 Msodium chloride, pH 5.5, may also be used. Alternatively, using methodsknown in the art, the cation exchange matrix may be equilibrated usingseveral column volumes of a dilute, weak base.

Alternatively, substantially purified hPP or hA or hFc may be eluted bycontacting the cation exchanger matrix with a buffer having asufficiently low pH or ionic strength to displace the hPP or hA from thematrix. The pH of the elution buffer may range from about pH 2.5 toabout pH 6.0. More specifically, the pH of the elution buffer may rangefrom about pH 2.5 to about pH 5.5, about pH 2.5 to about pH 5.0. Theelution buffer may have a pH of about 3.0. In addition, the quantity ofelution buffer may vary widely and will generally be in the range ofabout 2 to about 10 column volumes.

Following adsorption of the hPP or hA or hFc polypeptide to the cationexchanger matrix, substantially purified hPP or hA or hFc polypeptidemay be eluted by contacting the matrix with a buffer having asufficiently high pH or ionic strength to displace the hPP or hA or hFcpolypeptide from the matrix. Suitable buffers for use in high pH elutionof substantially purified hPP or hA or hFc polypeptide may include, butnot limited to, citrate, phosphate, formate, acetate, HEPES, and MESbuffers ranging in concentration from at least about 5 mM to at leastabout 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the hPP or hApolypeptide to isolate substantially purified hPP or hA or hFcpolypeptide. In this regard, silica derivatized resins with alkylfunctionalities with a wide variety of lengths, including, but notlimited to, at least about C₃ to at least about C₃₀, at least about C₃to at least about C₂₀, or at least about C₃ to at least about C₁₈,resins may be used. Alternatively, a polymeric resin may be used. Forexample, TosoHaas Amberchrome CG1000sd resin may be used, which is astyrene polymer resin. Cyano or polymeric resins with a wide variety ofalkyl chain lengths may also be used. Furthermore, the RP-HPLC columnmay be washed with a solvent such as ethanol. The Source RP column isanother example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the hPP or hA or hFc polypeptide from theRP-HPLC column. The most commonly used ion pairing agents include, butare not limited to, acetic acid, formic acid, perchloric acid,phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid,triethylamine, tetramethylammonium, tetrabutylammonium, andtriethylammonium acetate. Elution may be performed using one or moregradients or isocratic conditions, with gradient conditions preferred toreduce the separation time and to decrease peak width. Another methodinvolves the use of two gradients with different solvent concentrationranges. Examples of suitable elution buffers for use herein may include,but are not limited to, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on the hPPor hA pol ypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHYHANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, AmershamBiosciences (Piscataway, N.J.) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the hPP or hA or hFc polypeptide, the column maythen washed using standard buffers and conditions to remove unwantedmaterials but retaining the hPP or hA or hFc polypeptide on the HICcolumn. The hPP or hA or hFc polypeptide may be eluted with about 3 toabout 10 column volumes of a standard buffer, such as a HEPES buffercontaining EDTA and lower ammonium sulfate concentration than theequilibrating buffer, or an acetic acid/sodium chloride buffer, amongothers. A decreasing linear salt gradient using, for example, a gradientof potassium phosphate, may also be used to elute the hPP or hA or hFcmolecules. The eluant may then be concentrated, for example, byfiltration such as diafiltration or ultrafiltration. Diafiltration maybe utilized to remove the salt used to elute the hPP or hA or hFcpolypeptide.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first hPP or hA or hFc polypeptide mixture or anysubsequent mixture thereof, to remove any excess salts and to replacethe buffer with a suitable buffer for the next isolation step or evenformulation of the final drug product.

The non-naturally encoded amino acid present in the hPP or hA or hFcmolecule may also be utilized to provide separation from other cellularproteins that do not contain the non-naturally encoded amino acid. Sincethe non-naturally encoded amino acid may comprise unique chemicalfunctional groups, the coupling of the unique functional group toanother molecule may provide a substantial purification step. Forexample, the non-naturally encoded amino acid may be coupled to anothermolecule that facilitates separation from other proteins. Such moleculesfor coupling to the non-natural amino acid include, but are not limitedto, PEG and other polymers, beads, and other solid substrates.

The yield of hPP or hA or hFc polypeptide, including substantiallypurified hPP or hA or hFc polypeptide, may be monitored at each stepdescribed herein using techniques known to those of ordinary skill inthe art. Such techniques may also be used to assess the yield ofsubstantially purified hPP or hA or hFc polypeptide following the lastisolation step. For example, the yield of hPP or hA or hFc polypeptidemay be monitored using any of several reverse phase high pressure liquidchromatography columns, having a variety of alkyl chain lengths such ascyano RP-HPLC, C₁₈RP-HPLC; as well as cation exchange HPLC and gelfiltration HPLC.

In specific embodiments of the present invention, the yield of hPP or hAor hFc after each purification step may be at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, at leastabout 99.9%, or at least about 99.99%, of the hPP or hA or hFc in thestarting material for each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring hPP or hA or hFc polypeptide using Western blot and ELISAassays. For example, polyclonal antibodies may be generated againstproteins isolated from negative control yeast fermentation and thecation exchange recovery. The antibodies may also be used to probe forthe presence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of hPP or hA orhFc polypeptide from the proteinaceous impurities is based ondifferences in the strength of hydrophobic interactions. Elution isperformed with an acetonitrile gradient in diluted trifluoroacetic acid.Preparative HPLC is performed using a stainless steel column (filledwith 2.8 to 3.2 liter of Vydac C4 silicagel). The HydroxyapatiteUltrogel eluate is acidified by adding trifluoroacetic acid and loadedonto the Vydac C4 column. For washing and elution an acetonitrilegradient in diluted trifluoroacetic acid is used. Fractions arecollected and immediately neutralized with phosphate buffer. The hPP orhA or hFc polypeptide fractions which are within the IPC limits arepooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of hPP or hA or hFc polypeptide to the DEAE groups ismediated by ionic interactions. Acetonitrile and trifluoroacetic acidpass through the column without being retained. After these substanceshave been washed off, trace impurities are removed by washing the columnwith acetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and hPP or hA or hFc polypeptide is eluted with abuffer with increased ionic strength. The column is packed with DEAESepharose fast flow. The column volume is adjusted to assure an hPP orhA polypeptide load in the range of 3-10 mg hPP or hA or hFcpolypeptide/ml gel. The column is washed with water and equilibrationbuffer (sodium/potassium phosphate). The pooled fractions of the HPLCeluate are loaded and the column is washed with equilibration buffer.Then the column is washed with washing buffer (sodium acetate buffer)followed by washing with equilibration buffer. Subsequently, hPP or hAor hFc polypeptide is eluted from the column with elution buffer (sodiumchloride, sodium/potassium phosphate) and collected in a single fractionin accordance with the master elution profile. The eluate of the DEAESepharose column is adjusted to the specified conductivity. Theresulting drug substance is sterile filtered into Teflon bottles andstored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.The Endosafe™-PTS assay is a colorimetric, single tube system thatutilizes cartridges preloaded with LAL reagent, chromogenic substrate,and control standard endotoxin along with a handheld spectrophotometer.Alternate methods include, but are not limited to, a Kinetic LAL methodthat is turbidmetric and uses a 96 well format.

A wide variety of methods and procedures can be used to assess the yieldand purity of an hPP or hA protein comprising one or more non-naturallyencoded amino acids, including but not limited to, the Bradford assay,SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massspectrometry (including but not limited to, MALDI-TOF) and other methodsfor characterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

VIII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe hPP or hA or hFc polypeptides of the present invention.Derivatization of amino acids with reactive side-chains such as Lys, Cysand Tyr resulted in the conversion of lysine to N²-acetyl-lysine.Chemical synthesis also provides a straightforward method to incorporateunnatural amino acids. With the recent development of enzymatic ligationand native chemical ligation of peptide fragments, it is possible tomake larger proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu.Rev. Biochem, 69:923 (2000). Chemical peptide ligation and nativechemical ligation are described in U.S. Pat. No. 6,184,344, U.S. PatentPublication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046,WO 02/098902, and WO 03/042235, which are incorporated by referenceherein. A general in vitro biosynthetic method in which a suppressortRNA chemically acylated with the desired unnatural amino acid is addedto an in vitro extract capable of supporting protein biosynthesis, hasbeen used to site-specifically incorporate over 100 unnatural aminoacids into a variety of proteins of virtually any size. See, e.g., V. W.Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995,34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction. 14221 An in vivo method, termed selective pressureincorporation, was developed to exploit the promiscuity of wild-typesynthetases. See, e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F.M. Dong, L. Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophicstrain, in which the relevant metabolic pathway supplying the cell witha particular natural amino acid is switched off, is grown in minimalmedia containing limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin,J. Am Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. AmChem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).14281 Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995). 14291 For example, a suppressor tRNA wasprepared that recognized the stop codon UAG and was chemicallyaminoacylated with an unnatural amino acid. Conventional site-directedmutagenesis was used to introduce the stop codon TAG, at the site ofinterest in the protein gene. See, e.g., Sayers, J. R., Schmidt, W.Eckstein, F. 5′-3′ Exonucleases in phosphorothioate-basedolignoucleotide-directed mutagensis, Nucleic Acids Res, 16(3):791-802(1988). When the acylated suppressor tRNA and the mutant gene werecombined in an in vitro transcription/translation system, the unnaturalamino acid was incorporated in response to the UAG codon which gave aprotein containing that amino acid at the specified position.Experiments using [³H]-Phe and experiments with α-hydroxy acidsdemonstrated that only the desired amino acid is incorporated at theposition specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Stephan in Scientist 2005 Oct. 10; pages 30-33 describes additionalmethods to incorporate non-naturally encoded amino acids into proteins.Lu et al. in Mol Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of an hPP or hA or hFcpolynucleotide of the present invention using a cell-free (in-vitro)translational system. Translation systems may be cellular or cell-free,and may be prokaryotic or eukaryotic. Cellular translation systemsinclude, but are not limited to, whole cell preparations such aspermeabilized cells or cell cultures wherein a desired nucleic acidsequence can be transcribed to mRNA and the mRNA translated. Cell-freetranslation systems are commercially available and many different typesand systems are well-known. Examples of cell-free systems include, butare not limited to, prokaryotic lysates such as Escherichia colilysates, and eukaryotic lysates such as wheat germ extracts, insect celllysates, rabbit reticulocyte lysates, rabbit oocyte lysates and humancell lysates. Eukaryotic extracts or lysates may be preferred when theresulting protein is glycosylated, phosphorylated or otherwise modifiedbecause many such modifications are only possible in eukaryotic systems.Some of these extracts and lysates are available commercially (Promega;Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; ArlingtonHeights, Ill.; GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, suchas the canine pancreatic extracts containing microsomal membranes, arealso available which are useful for translating secretory proteins. Inthese systems, which can include either mRNA as a template (in-vitrotranslation) or DNA as a template (combined in-vitro transcription andtranslation), the in vitro synthesis is directed by the ribosomes.Considerable effort has been applied to the development of cell-freeprotein expression systems. See, e.g., Kim, D. M. and J. R. Swartz,Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D. M. and J.R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., andJ. R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M.,and J. R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999);and Patnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998);U.S. Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of hPP or hA orhFc polypeptides comprising a non-naturally encoded amino acid includesthe mRNA-peptide fusion technique. See, e.g., R. Roberts and J. Szostak,Proc. Natl Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al.,Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAtemplate linked to puromycin is translated into peptide on the ribosome.If one or more tRNA molecules has been modified, non-natural amino acidscan be incorporated into the peptide as well. After the last mRNA codonhas been read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of hPP or hA or hFcpolypeptides comprising one or more non-naturally encoded amino acids toidentify polypeptides having desired properties. More recently, in vitroribosome translations with purified components have been reported thatpermit the synthesis of peptides substituted with non-naturally encodedamino acids. See, e.g., A. Forster et al., Proc. Natl Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

IX. Macromolecular Polymers Coupled to hPP or hA or hFc Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to hPP or hA polypeptides of the present invention to modulatebiological properties of the hPP or hA polypeptide, and/or provide newbiological properties to the hPP or hA or hFc molecule. Thesemacromolecular polymers can be linked to the hPP or hA or hFcpolypeptide via a naturally encoded amino acid, via a non-naturallyencoded amino acid, or any functional substituent of a natural ornon-natural amino acid, or any substituent or functional group added toa natural or non-natural amino acid. The molecular weight of the polymermay be of a wide range, including but not limited to, between about 100Da and about 100,000 Da or more. The molecular weight of the polymer maybe between about 100 Da and about 100,000 Da, including but not limitedto, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da,70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated hPP or hA or hFc polypeptide preparations provided herein arethose which are homogenous enough to display the advantages of ahomogenous preparation, e.g., ease in clinical application inpredictability of lot to lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:hPP or hA or hFc polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount willvary from one individual to another and will depend upon a number offactors, including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of hPP or hAor hFc polypeptide used for therapy gives an acceptable rate of changeand maintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the hPP or hAor hFc polypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to an hPP or hA or hFc polypeptide via a naturally-occurringor non-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the hPP or hA or hFc polypeptide to form a Huisgen[3+2] cycloaddition product. Alternatively, an alkyne group on the PEGcan be reacted with an azide group present in a non-naturally encodedamino acid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into the hPPor hA or hFc polypeptide via a non-naturally encoded amino acid and usedto react preferentially with a ketone or aldehyde group present in thewater soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG isbetween about 100 Da and 50,000 Da. In some embodiments, PEG is betweenabout 100 Da and 40,000 Da. In some embodiments, PEG is between about1,000 Da and 40,000 Da. In some embodiments, PEG is between about 5,000Da and 40,000 Da. In some embodiments, PEG is between about 10,000 Daand 40,000 Da. Branched chain PEGs, including but not limited to, PEGmolecules with each chain having a MW ranging from 1-100 kDa (includingbut not limited to, 1-50 kDa or 5-20 kDa) can also be used. Themolecular weight of each chain of the branched chain PEG may be,including but not limited to, between about 1,000 Da and about 100,000Da or more. The molecular weight of each chain of the branched chain PEGmay be between about 1,000 Da and about 100,000 Da, including but notlimited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 1,000 Daand 50,000 Da. In some embodiments, the molecular weight of each chainof the branched chain PEG is between about 1,000 Da and 40,000 Da. Insome embodiments, the molecular weight of each chain of the branchedchain PEG is between about 5,000 Da and 40,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 5,000 Da and 20,000 Da. A wide range of PEG molecules aredescribed in, including but not limited to, the Shearwater Polymers,Inc. catalog, Nektar Therapeutics catalog, incorporated herein byreference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the hPP or hA or hFc polypeptide variant with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof PEG is between about 5,000 Da and 40,000 Da. In some embodiments, themolecular weight of PEG is between about 10,000 Da and 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about1,000 Da and 40,000 Da. In some embodiments, the molecular weight ofeach chain of the polymer backbone is between about 5,000 Da and 40,000Da. In some embodiments, the molecular weight of each chain of thepolymer backbone is between about 10,000 Da and 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B—N═N═N

wherein:

-   N═N═N is an azide moiety;-   B is a linking moiety, which may be present or absent;-   POLY is a water-soluble non-antigenic polymer;-   A is a linking moiety, which may be present or absent and which may    be the same as B or different; and-   X is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. JBiochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Biotechnology (NY)8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem.4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N

wherein:

-   X is a functional group as described above; and-   n is about 20 to about 4000.

In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═N

wherein:

-   W is an aliphatic or aromatic linker moiety comprising between 1-10    carbon atoms;-   n is about 20 to about 4000; and-   X is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.

X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N

wherein:

-   PEG is poly(ethylene glycol) and X is a capping group such as alkoxy    or a functional group as described above; and-   M is a functional group that is not reactive with the azide    functionality but that will react efficiently and selectively with    the N functional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:

BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:

X-A-POLY-B—C≡C—R

wherein:

-   R can be either H or an alkyl, alkene, alkyoxy, or aryl or    substituted aryl group;-   B is a linking moiety, which may be present or absent;-   POLY is a water-soluble non-antigenic polymer;-   A is a linking moiety, which may be present or absent and which may    be the same as B or different; and-   X is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:

-   X is a functional group as described above;-   n is about 20 to about 4000; and-   m is between 1 and 10.    Specific examples of each of the heterobifunctional PEG polymers are    shown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+-C≡CR′→X-PEG-C≡CR′

wherein:

-   PEG is poly(ethylene glycol) and X is a capping group such as alkoxy    or a functional group as described above; and-   R′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a    substituted alkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the hPP or hA or hFcpolypeptides of the invention. The water soluble polymers may be linkedvia a non-naturally encoded amino acid incorporated in the hPP or hA orhFc polypeptide or any functional group or substituent of anon-naturally encoded or naturally encoded amino acid, or any functionalgroup or substituent added to a non-naturally encoded or naturallyencoded amino acid. Alternatively, the water soluble polymers are linkedto an hPP or hA or hFc polypeptide incorporating a non-naturally encodedamino acid via a naturally-occurring amino acid (including but notlimited to, cysteine, lysine or the amine group of the N-terminalresidue). In some cases, the hPP or hA or hFc polypeptides of theinvention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural aminoacids, wherein one or more non-naturally-encoded amino acid(s) arelinked to water soluble polymer(s) (including but not limited to, PEGand/or oligosaccharides). In some cases, the hPP or hA or hFcpolypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more naturally-encoded amino acid(s) linked to water solublepolymers. In some cases, the hPP or hA or hFc polypeptides of theinvention comprise one or more non-naturally encoded amino acid(s)linked to water soluble polymers and one or more naturally-occurringamino acids linked to water soluble polymers. In some embodiments, thewater soluble polymers used in the present invention enhance the serumhalf-life of the hPP or hA or hFc polypeptide relative to theunconjugated form.

The number of water soluble polymers linked to an hPP or hA or hFcpolypeptide (i.e., the extent of PEGylation or glycosylation) of thepresent invention can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of hPP or hA or hFc is increased at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold,30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over anunmodified polypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., hydrazide,hydrazine, hydroxylamine or semicarbazide)

In one embodiment of the present invention, an hPP or hA or hFcpolypeptide comprising a carbonyl-containing non-naturally encoded aminoacid is modified with a PEG derivative that contains a terminalhydrazine, hydroxylamine, hydrazide or semicarbazide moiety that islinked directly to the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising a carbonyl-containing amino acid is modified with a PEGderivative that contains a terminal hydroxylamine, hydrazide, hydrazine,or semicarbazide moiety that is linked to the PEG backbone by means ofan amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising a carbonyl-containing amino acid is modified with a branchedPEG derivative that contains a terminal hydrazine, hydroxylamine,hydrazide or semicarbazide moiety, with each chain of the branched PEGhaving a MW ranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising a non-naturally encoded amino acid is modified with a PEGderivative having a branched structure. For instance, in someembodiments, the hydrazine- or hydrazide-terminal PEG derivative willhave the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe hPP or hA polypeptide can modulate the binding of the hPP or hA orhFc polypeptide to the hPP or hA or hFc polypeptide receptor or bindingpartner. In some embodiments, the linkages are arranged such that thehPP or hA or hFc polypeptide binds the hPP or hA polypeptide receptor orbinding partner with a K_(d) of about 400 nM or lower, with a K_(d) of150 nM or lower, and in some cases with a K_(d) of 100 nM or lower, asmeasured by an equilibrium binding assay, such as that described inSpencer et al., J. Biol. Chem., 263:7862-7867 (1988).

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of hPP or hA orhFc polypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, hPP or hA or hFc polypeptide is PEGylated with analkyne-terminated mPEG derivative. Briefly, an excess of solidmPEG(5000)-O—CH₂—C≡CH is added, with stirring, to an aqueous solution ofp-azido-L-Phe-containing hPP or hA or hFc polypeptide at roomtemperature. Typically, the aqueous solution is buffered with a bufferhaving a pK_(a) near the pH at which the reaction is to be carried out(generally about pH 4-10). Examples of suitable buffers for PEGylationat pH 7.5, for instance, include, but are not limited to, HEPES,phosphate, borate, TRIS-HCl, EPPS, and TES. The pH is continuouslymonitored and adjusted if necessary. The reaction is typically allowedto continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated hPP or hA or hFcpolypeptide variants from free mPEG(5000)-O—CH₂—C≡CH and anyhigh-molecular weight complexes of the pegylated hPP or hA or hFcpolypeptide which may form when unblocked PEG is activated at both endsof the molecule, thereby crosslinking hPP or hA or hFc polypeptidevariant molecules. The conditions during hydrophobic interactionchromatography are such that free mPEG(5000)-O—CH₂—C≡CH flows throughthe column, while any crosslinked PEGylated hPP or hA or hFc polypeptidevariant complexes elute after the desired forms, which contain one hPPor hA or hFc polypeptide variant molecule conjugated to one or more PEGgroups. Suitable conditions vary depending on the relative sizes of thecross-linked complexes versus the desired conjugates and are readilydetermined by those of ordinary skill in the art. The eluent containingthe desired conjugates is concentrated by ultrafiltration and desaltedby diafiltration.

If necessary, the PEGylated hPP or hA or hFc polypeptide obtained fromthe hydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the hPP or hA-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky R B., et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of an hPP or hA or hFcpolypeptide of the invention can be further derivatized or substitutedwithout limitation.

Azide-Containing PEG Derivatives

In another embodiment of the invention, an hPP or hA or hFc polypeptideis modified with a PEG derivative that contains an azide moiety thatwill react with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising a alkyne-containing amino acid is modified with a branchedPEG derivative that contains a terminal azide moiety, with each chain ofthe branched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.

Alkyne-Containing PEG Derivatives

In another embodiment of the invention, an hPP or hA or hFc polypeptideis modified with a PEG derivative that contains an alkyne moiety thatwill react with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising an alkyne-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal azide orterminal alkyne moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, an hPP or hA or hFc polypeptidecomprising an azide-containing amino acid is modified with a branchedPEG derivative that contains a terminal alkyne moiety, with each chainof the branched PEG having a MW ranging from 10-40 kDa and may be from5-20 kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.

Phosphine-Containing PEG Derivatives

In another embodiment of the invention, an hPP or hA or hFc polypeptideis modified with a PEG derivative that contains an activated functionalgroup (including but not limited to, ester, carbonate) furthercomprising an aryl phosphine group that will react with an azide moietypresent on the side chain of the non-naturally encoded amino acid. Ingeneral, the PEG derivatives will have an average molecular weightranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to hPP or hA or hFcpolypeptides, as well as PEGylation methods include those described in,e.g., U.S. Patent Publication No. 2004/0001838; 2002/0052009;2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333;2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023;2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573;2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171;2001/0044526; 2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778;5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502; 5,281,698;5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167; 6,610,281;6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237; 5,900,461;5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460; 5,324,844;5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821; 5,559,213;5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,912; WO97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758,WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924,WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO95/13312, EP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670,EP 605 963, EP 510 356, EP 400 472, EP 183 503 and EP 154 316, which areincorporated by reference herein. Any of the PEG molecules describedherein may be used in any form, including but not limited to, singlechain, branched chain, multiarm chain, single functional, bi-functional,multi-functional, or any combination thereof.

Enhancing Affinity of Biologically Active Molecules for hA

Various biologically active molecules can also be fused to the hApolypeptides of the invention to modulate the half-life of thebiologically active molecule, or modulate another property of thebiologically active molecule. In some embodiments, biologically activemolecules are linked or fused to hA polypeptides of the invention toenhance affinity for endogenous binding partners.

For example, in some cases, a recombinant fusion of a biologicallyactive molecule and hA is made. Exemplary albumin binding sequencesinclude, but are not limited to, the albumin binding domain fromstreptococcal protein G (see. e.g., Makrides et al., J. Pharmacol. Exp.Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In other embodiments, the biologically active molecule of the presentinvention are acylated with fatty acids. In some cases, the fatty acidspromote binding to hA. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the biologically active molecule polypeptides ofthe invention are fused directly with hA. Those of skill in the art willrecognize that a wide variety of other biologically active molecules canalso be linked to other hPP's in the present invention to modulatebinding to binding partners.

X. Glycosylation of hPP or hA or hFc Polypeptides

The invention includes hPP or hA or hFc polypeptides incorporating oneor more non-naturally encoded amino acids bearing saccharide residues.The saccharide residues may be either natural (including but not limitedto, N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N— or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C— or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to hPP or hA or hFc polypeptides either in vivo or in vitro. Insome embodiments of the invention, an hPP or hA or hFc polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a saccharide derivatized with an aminooxy group togenerate the corresponding glycosylated polypeptide linked via an oximelinkage. Once attached to the non-naturally encoded amino acid, thesaccharide may be further elaborated by treatment withglycosyltransferases and other enzymes to generate an oligosaccharidebound to the hPP or hA or hFc polypeptide. See, e.g., H. Liu, et al. J.Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, an hPP or hA or hFc polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified directly with a glycan with defined structure prepared as anaminooxy derivative. One of ordinary skill in the art will recognizethat other functionalities, including azide, alkyne, hydrazide,hydrazine, and semicarbazide, can be used to link the saccharide to thenon-naturally encoded amino acid.

In some embodiments of the invention, an hPP or hA or hFc polypeptidecomprising an azide or alkynyl-containing non-naturally encoded aminoacid can then be modified by, including but not limited to, a Huisgen[3+2] cycloaddition reaction with, including but not limited to, alkynylor azide derivatives, respectively. This method allows for proteins tobe modified with extremely high selectivity.

XL Measurement of Biologically Active Molecule Activity

Regardless of which methods are used to create the hPP or hA or hFc,they are subject to assays for biological activity. Tritiated thymidineassays may be conducted to ascertain the degree of cell division, ifappropriate. Other biological assays, however, may be used to ascertainthe desired activity. Biological assays such as measuring the ability toinhibit an antigen's biological activity, such as an enzymatic,proliferative, or metabolic activity also provides an indication of hPPor hFc activity. Other in vitro assays may be used to ascertainbiological activity. In general, the test for biological activity shouldprovide analysis for the desired result, such as increase or decrease inbiological activity (as compared to non-altered hPP or hFc), differentbiological activity (as compared to non-altered hPP or hFc), receptor orbinding partner affinity analysis, conformational or structural changesof the hPP or hFc itself or binding partner (as compared to thenon-altered hPP or hFc), or serum half-life analysis, as appropriate forthe antigen's biological activity.

The above compilation of references for assay methodologies is notexhaustive, and those of ordinary skill in the art will recognize otherassays useful for testing for the desired end result.

XIII. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the hPP or hA or hFcpolypeptide with or without conjugation of the polypeptide to a watersoluble polymer moiety. The rapid decrease of hPP or hA or hFcpolypeptide serum concentrations has made it important to evaluatebiological responses to treatment with conjugated and non-conjugated hPPor hA or hFc polypeptide and variants thereof. The conjugated andnon-conjugated hPP or hA or hFc polypeptide and variants thereof of thepresent invention may have prolonged serum half-lives also aftersubcutaneous or i.v. administration, making it possible to measure by,e.g. ELISA method or by a primary screening assay. ELISA or RIA kitsfrom either BioSource International (Camarillo, Calif.) or DiagnosticSystems Laboratories (Webster, Tex.) may be used. Measurement of in vivobiological half-life is carried out as described herein.

The potency and functional in vivo half-life of an hPP or hA or hFcpolypeptide comprising a non-naturally encoded amino acid can bedetermined according to the protocol described in Clark, R., et al., J.Biol. Chem. 271(36): 21969-21977 (1996).

Pharmacokinetic parameters for an hPP or hA or hFc polypeptidecomprising a non-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for an hPP or hA or hFcpolypeptide comprising a non-naturally encoded amino acid not conjugatedto a water soluble polymer and about 4 days for an hPP or hA polypeptidecomprising a non-naturally encoded amino acid and conjugated to a watersoluble polymer. Pharmacokinetic data for hPP or hA or hFc polypeptidesis well-studied in several species and can be compared directly to thedata obtained for hPP or hA or hFc polypeptides comprising anon-naturally encoded amino acid. See Mordenti J., et al., Pharm. Res.8(11):1351-59 (1991) for studies related to hPP or hA or hFc.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum hPP or hA or hFc levels aremonitored over time.

The specific activity of hPP or hA or hFc polypeptides in accordancewith this invention can be determined by various assays known in theart. The biological activity of the hPP or hA or hFc polypeptidemuteins, or fragments thereof, obtained and purified in accordance withthis invention can be tested by methods described or referenced hereinor known to those of ordinary skill in the art.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, hPP or hA or hFc, synthetases, proteins comprising one or moreunnatural amino acid, etc.) are optionally employed for therapeuticuses, including but not limited to, in combination with a suitablepharmaceutical carrier. Such compositions, for example, comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier or excipient includes,but is not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and/or combinations thereof. The formulation is madeto suit the mode of administration. In general, methods of administeringproteins are known to those of ordinary skill in the art and can beapplied to administration of the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of an hPP or hA or hFc polypeptide modified toinclude one or more unnatural amino acids to a natural amino acid hPP orhA or hFc polypeptide), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

hPP or hA or hFc polypeptides of the invention may be administered byany conventional route suitable for proteins or peptides, including, butnot limited to parenterally, e.g. injections including, but not limitedto, subcutaneously or intravenously or any other form of injections orinfusions. Polypeptide compositions can be administered by a number ofroutes including, but not limited to oral, intravenous, intraperitoneal,intramuscular, ophthalmic, intraocular, intracranial, subdural, into theCSF, transdermal, subcutaneous, topical, sublingual, or rectal means.Compositions comprising non-natural amino acid polypeptides, modified orunmodified, can also be administered via liposomes. Such administrationroutes and appropriate formulations are generally known to those ofskill in the art. The hPP or hA or hFc polypeptide comprising anon-naturally encoded amino acid, may be used alone or in combinationwith other suitable components such as a pharmaceutical carrier. The hPPor hA or hFc polypeptide comprising a non-naturally encoded amino acid,may also be used in combination with a pharmaceutical carrier that isbiodegradable or biosoluble for modulated release or availability of theactive agent.

The hPP or hA or hFc polypeptide comprising a non-natural amino acid,alone or in combination with other suitable components, can also be madeinto aerosol formulations (i.e., they can be “nebulized”) to beadministered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intraocular, intracranial, andsubcutaneous routes, include aqueous and non-aqueous, isotonic sterileinjection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations of hPP or hA orhFc can be presented in unit-dose or multi-dose sealed containers, suchas ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for albumin, albumin fusionswith other polypeptides, EPO, GH, G-CSF, GM-CSF, IFNs, interleukins,antibodies, antibody fragments, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, depending on the application. The dose isdetermined by the efficacy of the particular vector, or formulation, andthe activity, stability or serum half-life of the unnatural amino acidpolypeptide employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, formulation, or the like in a particular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

The hPP or hA or hFc polypeptides of the invention can be administereddirectly to a mammalian subject. Administration is by any of the routesnormally used for introducing hPP or hA or hFc polypeptide to a subject.The hPP or hA or hFc polypeptide compositions according to embodimentsof the present invention include those suitable for oral, rectal,topical, inhalation (including but not limited to, via an aerosol),buccal (including but not limited to, sublingual), vaginal, parenteral(including but not limited to, subcutaneous, intramuscular, intradermal,intraarticular, intrapleural, intraperitoneal, inracerebral,intraarterial, or intravenous), topical (i.e., both skin and mucosalsurfaces, including airway surfaces) and transdermal administration,although the most suitable route in any given case will depend on thenature and severity of the condition being treated. Administration canbe either local or systemic. The formulations of compounds can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials. The hPP or hA or hFc polypeptides of the invention can beprepared in a mixture in a unit dosage injectable form (including butnot limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier. The hPP or hA or hFc polypeptidesof the invention can also be administered by continuous infusion (using,including but not limited to, minipumps such as osmotic pumps), singlebolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions and formulations of the invention maycomprise a pharmaceutically acceptable carrier, excipient, orstabilizer. Pharmaceutically acceptable carriers are determined in partby the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions (including optional pharmaceutically acceptable carriers,excipients, or stabilizers) of the present invention (see, e.g.,Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

Suitable carriers include but are not limited to, buffers containingsuccinate, phosphate, borate, HEPES, citrate, histidine, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA and edentate disodium; divalent metal ions includingbut not limited to, zinc, cobalt, or copper; sugar alcohols includingbut not limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium and sodium chloride; and/ornonionic surfactants including but not limited to Tween™ (including butnot limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20),Pluronics™ and other pluronic acids, including but not limited to, andother pluronic acids, including but not limited to, pluronic acid F68(poloxamer 188), or PEG. Suitable surfactants include for example butare not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R-Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated hPP or hA or hFc against one or more stresses including butnot limited to stress that results from agitation. Some of the above maybe referred to as “bulking agents.” Some may also be referred to as“tonicity modifiers.” Antimicrobial preservatives may also be appliedfor product stability and antimicrobial effectiveness; suitablepreservatives include but are not limited to, benzyl alcohol,benzalkonium chloride, metacresol, methyl/propyl parabene, cresol, andphenol, or a combination thereof.

hPP or hA or hFc polypeptides of the invention, including those linkedto water soluble polymers such as PEG can also be administered by or aspart of sustained-release systems. Sustained-release compositionsinclude, including but not limited to, semi-permeable polymer matricesin the form of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA., 77: 4030-4034 (1980); EP 52,322; EP 36,676;U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; JapanesePat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. All references and patents cited are incorporated by referenceherein.

Liposomally entrapped hPP or hA or hFc polypeptides can be prepared bymethods described in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl.Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the hPP or hA or hFc polypeptide of the present inventionadministered parenterally per dose is in the range of about 0.01μg/kg/day to about 100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, ofpatient body weight, although this is subject to therapeutic discretion.The frequency of dosing is also subject to therapeutic discretion, andmay be more frequent or less frequent than the commercially availablehPP or hA or hFc polypeptide products approved for use in humans.Generally, a PEGylated hPP or hA or hFc polypeptide of the invention canbe administered by any of the routes of administration described above.

Examples

The following examples are offered to illustrate, but do not to limitthe claimed invention.

Example 1

This example describes one of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into hA. Using the criteria described below, theamino acid positions utilized for site-specific incorporation ofp-acetyl-phenylalanine (pAF) into HSA are NO: 34, 82, 172, 301, 364,505. Several HSA crystal structures were used to determine preferredpositions into which one or more non-naturally encoded amino acids couldbe introduced: the coordinates for these structures are available fromthe Protein Data Bank (PDB) via The Research Collaboratory forStructural Bioinformatics at www.rcsb.org (PDB IDs 1A06, 1E78 and 1BMO).X-ray crystal structure information was used to perform solventaccessibility calculations on the HSA molecule, utilizing the Cx program(Pintar et al. Bioinformatics, 2002, Vol. 18, p 980). The solventaccessibility of all atoms was calculated and a composite Cx value foreach amino acid residue was determined, and is shown in FIG. 2. Aminoacids were rank-ordered by Cx value and correlated with their3-dimensional position in the HSA structure, and certain of these sitesare shown on FIG. 3. HSA contains a total of 582 amino acids: the top 51Cx values were examined for pAF substitution. Sites were chosen in orderto place pAF into solvent-exposed regions of the HSA structure wherecovalent conjugation would be most feasible. The following criteria wereused to evaluate the top 51 Cx positions of HSA for the introduction ofa non-naturally encoded amino acid: the selected residues (a) shouldhave a maximal Cx value, demonstrating solvent-accessibility and minimalvan der Waals or hydrogen bonding interactions with surroundingresidues, b) should be from different surface-exposed regions of theprotein and c) should be in areas of both rigid and flexible proteinstructure. A partial listing of amino acid positions suitable forincorporation of non-naturally encoded amino acids into hA are shown inFIG. 4.

Example 2

This example details cloning and expression of a hA polypeptide with andwithout a non-naturally encoded amino acid in yeast. [Cloning of thealbumin DNA into expression vector, transformation of yeast]

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress hA containing a non-naturally encoded amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into hA, in response to an encoded selector codon.

TABLE 2 hA, O-RS and O-tRNA sequences. SEQ ID NO: 1 Human albumin aminoacid sequence hA SEQ ID NO: 2 Nucleotide sequence encoding human albuminhA SEQ ID NO: 3 M. jannaschii mtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 4HLAD03; an optimized amber supressor tRNA tRNA SEQ ID NO: 5 HL325A; anoptimized AGGA frameshift supressor tRNA tRNA SEQ ID NO: 6 AminoacyltRNA synthetase for the incorporation of p-azido-L-phenylalanine RSp-Az-PheRS(6) SEQ ID NO: 7 Aminoacyl tRNA synthetase for theincorporation of p-benzoyl-L-phenylalanine RS p-BpaRS(1) SEQ ID NO: 8Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 9 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalanine RSPropargyl-PheRS SEQ ID NO: 10 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO:11 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine RS p-Az-PheRS(1) SEQ ID NO: 12 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine RSp-Az-PheRS(3) SEQ ID NO: 13 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(4) SEQ ID NO: 14Aminoacyl tRNA synthetase for the incorporation of p-azido-phenylalanineRS p-Az-PheRS(2) SEQ ID NO: 15 Aminoacyl tRNA synthetase for theincorporation of p-acetyl-phenylalanine (LW1) RS SEQ ID NO: 16 AminoacyltRNA synthetase for the incorporation of p-acetyl-phenylalanine (LW5) RSSEQ ID NO: 17 Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW6) RS SEQ ID NO: 18 Aminoacyl tRNA synthetasefor the incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO:19 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine (AzPheRS-6) RS

The transformation of yeast with plasmids containing the modified hAgene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specificfor the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into thehA polypeptide. The transformed yeast, grown at 37° C. in mediacontaining between 0.01-100 mM of the particular non-naturally encodedamino acid, expresses modified hA with high fidelity and efficiency.

Saccharomyces cerevisiae strain MaV203 carrying either pGADHSA orpGADGAL4(control) were cultured 23 hours in YPD. Cells and supernatantswere harvested by centrifugation at 4000 g for 5 minutes at 4° C. Cellextracts were generated by lysis of about 10 mg wet cell pellet.Supernatants were concentrated approximately 30× by 10 kDa MWCO spincolumns. 16 μl of reduced sample loaded into each well of a 4-12%Bis-Tris PAGE gel, run 50 minutes at 200V and either stained withCoomassie blue or transferred to nitrocellulose membrane (25V, 70minutes). Blot was probed with anti-HSA IgY pAb (200 ng/ml) primary andHRP-conjugated goat anti-IgY IgG (10 ng/ml), then detected usingSupersignal ECL substrate (Sigma) and Biorad Fluor-S imaging system. Theresults are shown in FIG. 5.

Methods for purification of hA are known to those of ordinary skill inthe art and are confirmed by SDS-PAGE, Western Blot analyses, orelectrospray-ionization ion trap mass spectrometry and the like.

Cloning of the Albumin DNA into Expression Vector, Transformation ofYeast

The wildtype hA coding sequence (CDS) was cloned into yeastshuttle/expression vector pGADGAL4 as follows. Commercially available hAcDNA was obtained and amplified by PCR using primers specific for the 5′and 3′ end of the hA CDS; HindIII restriction sites were integrated intothe primer ends. Following PCR, the correct fragment was digested withHindIII (37° C., 60 minutes), purified, and ligated to HindIII-cutpGADGAL4. Ligation products were transformed into TOP10 chemicallycompetent E. coli; plasmid DNA was isolated from selected transformantsusing a miniprep kit. Screening for the desired plasmid product wascarried out by independent EcoRV and PstI restriction digests of eachselected plasmid clone; those clones exhibiting the expected bandingpattern following agarose gel electrophoresis (130V, 30 minutes) andEtBr staining were verified by sequencing. Clones were considered‘positive’ if they had a copy of the hA CDS replacing the GAL4 region ofpGADGAL4; the specific plasmid to be used in further experiments wasnamed ‘pGADHSA’.

Amber mutations were made to the specified codons within the hA CDS bythe Quickchange method of site-directed mutagenesis (Stratagene, LaJolla, Calif.). Briefly, overlapping primers specific for the region tobe mutated and containing the nucleotide-base changes necessary togenerate it, were used in a PCR reaction to generate semi-mutated hybridplasmid DNA molecules. Following DpnI restriction digestion to cleaveand destroy the parental, methylated DNA strands, the products weretransformed into TOP10 chemically competent E. coli. Transformants wereselected; plasmid DNA was isolated and plasmid DNA containing thedesired mutations were confirmed by nucleotide sequencing.

Saccharomyces cerevisiae transformations were carried out according toprotocols detailed by R. D. Geitz(http://www.umanitoba.ca/faculties/medicine/biochem/gietz/Trafo.html).Briefly, freshly grown S. cerevisiae were scraped from YPD-agar platesin approximately 50 microliter aliquots, washed with sterile water, andresuspended in a transformation mix containing 33% poly(ethyleneglycol)-3350, 100 mM LiOAc, 300 microgram/ml single stranded salmonsperm DNA, and 5-10 micrograms transforming plasmid DNA). Cells wereheat-shocked for 40-60 minutes at 42° C., washed and resuspended in 1.0ml sterile water, and plated in dilutions onto selective agar plates.Tranformants were then used in subsequent expression and suppressionexperiments.

Expression and characterization of the HSA protein with non-naturalamino acid.

Saccharomyces cerevisiae strain InvSc1 was transformed with either:

-   -   1.) pGADHSA(amber) alone,    -   2.) pGADHSA(amber) plus plasmid containing the E. coli tyrosine        tRNA synthetase gene and the tRNA_(cuA) gene,    -   3.) pGADHSA(amber) plus plasmid containing the E. coli        para-acetylphenylalanine tRNA synthetase gene and the tRNA_(CUA)        gene.

Transformants were cultured in 50 ml SD media (lacking leucine, lackingtryptophan), and in the case of (3) above, in the presence of 1 mMpara-acetylphenylalanine, at 30° C. with shaking, to an OD₆₀₀ of 1.0. Atthis point, 30 OD₆₀₀ equivalents from each culture were pelleted bycentrifugation at 3000×g for 5 minutes and resuspended in 30 ml YPD(again, in the case of (3) above, also in the presence of 1 mMpara-acetylphenylalanine). Cultures were again allowed to grow at 30degrees C. at 250 rpm shaking for a further 24 hours. 50 OD₆₀₀equivalents from each culture were then pelleted as above; the culturesupernatant was isolated, and a fraction of it was concentrated roughly30-fold using a 10-kDa MWCO spin column.

The presence of hA protein was assayed by immunoblot, and is describedas follows. 16 μl of each reduced sample was loaded into each well of a4-12% Bis-Tris PAGE gel, run 50 minutes at 200V and either stained withCoomassie blue or transferred to nitrocellulose membrane (25V, 70minutes). Blot was probed with anti-HSA IgY pAb (200 ng/ml) primary andHRP-conjugated goat anti-IgY IgG (10 ng/ml), then detected usingSupersignal ECL substrate (Sigma) and Biorad Fluor-S imaging system. Theresults are shown in FIG. 6, and demonstrate incorporation of anon-naturally encoded amino acid into the hA polypeptide.

Non-Naturally Encoded amino Acid Suppression of HSA-C34

The S. cerevisiae Y187 strains transformed with plasmids encoding WTHSA, HSA-C34 or HSA-C34 plus a plasmid encoding a tRNA synthetase(RS)/tRNA pair [tyrosine (Y)RS, pAFRS, pAzRS, pBzRS, OMeRS] were grownin HC-Leu media or HC-Leu-Trp media overnight (30° C., 200 rpm). Cellswere pelleted at 5000×g for 5 min at 4° C. and resuspended in YPAD to anOD600 of 0.5. Strains containing an exogenous tRNA/RS pair wereincubated in the presence (+) or absence (−) of the appropriate novelamino acid (pAF, pAZ-Phe, pBz-Phe, OMe-Tyr, 1 mM).pAF=para-acetylphenylalanine; pAZ-Phe-para-azidophenylalanine;pBz-Phe=para-benzoyl-L-phenylalanine; OMe-Tyr=O-methyltyrosine.Following a 24 hour incubation (30° C., 200 rpm), all cultures wereharvested by centrifugation; 15 mL of reduced supernatant was resolvedby SDS-PAGE with a 4-12% Bis-Tris gel and visualized by (FIG. 10A)Coomassie and (FIGS. 10B and C) anti-HSA Western blot. HSA Std is 200 ngHSA purified from human serum.

Conjugation of HSA-C34-pAF to 5K amino-oxyPEG

Purified wt HSA or HSA-C34-pAF was buffer-exchanged into reaction buffer(20 mM sodium acetate, 20 g/L glycine, 5 g/L mannitol, 1 mM EDTA, pH4.0), at a final concentration of ˜2 mg/ml. Reactions on HSA-C34-pAFwere initiated either with the addition of 20 molar equivalents of 5Kamino-oxy derivatized PEG (+) or with the addition of reaction buffer(−); acetic hydrazide (catalyst: 50 mM final concentration) was added toall reactions. Reactions were allowed to proceed undisturbed at 28° C.for 48 hours. 1 μl of reaction mix per lane was then separated on a4-12% bis-tris polyacrylamide gel and analyzed by α-HSA western blot.See FIG. 11.

HSA Analytical Methods Trypsinization of Samples

WT HSA (Sigma-Aldrich, 2 mg/ml) or purified, pAF-suppressed HSA (˜2mg/ml) was diluted into 6M guanidine-HCl and 50 mM Tris pH 7.5 (finalconcentration). Samples were reduced with 20 mM DTT at 37° C. for 1 hourfollowed by alkylation with 40 mm iodoacetic acid (IAA) for 40 minutesat room temperature in the dark. The reaction was quenched with 40 mmDTT, and samples were dialyzed into 50 mM Tris, 1 mM CaCl2 pH 7.5 andtreated with trypsin 1:20 (enzyme:protein) for 4 hours at 3.7° C. Thereaction was quenched with addition of TFA to 0.1%.

LC-MS/MS:

100 μL of trypsinized sample was loaded onto a Zorbax SB-C18 column(2.1×150 mm 3.5 μm, 40° C.), at 0.2 mL/min. Peptides were eluted in0.05% TFA with a 1.38%/min gradient of 0 to 100% acetonitrile over 60minutes. Eluted peptides were electrosprayed directly onto aThermoElectron LCQ Deca with the application of 15V capillary voltageand 4.5 kV spray voltage. A cycle of one full-scan mass spectrum(300-2000 m/z) followed by data-dependent tandem MS spectra acquisitionwas performed at a 35% normalized collision energy throughout peptideelution

Peptide Mass Map of HSA-C34pAF

Purified wt HSA and HSA-C34pAF (approx. 60 μg) were subjected toproteolytic digestion with trypsin, and LC-MS/MS was performed onsamples with an LCQ Deca (Thermoelectron) to obtain a peptide mass map.Based on sequence analysis, the fifth tryptic peptide from theN-terminus (T5) in WT HSA contains Cys34; T5 in pAF-suppressed HSAshould exhibit a mass difference corresponding to the Cys34pAF aminoacid substitution. The WT T5 peptide (calculated M2H+m/z=1246.95) elutedfrom the reverse phase column at 37.3 minutes with an observed m/z of1246.4; the tryptic mass map of HSA-C34pAF at 37.3 minutes did not yielda peptide of m/z of 1246+/−2. Conversely, pAF-substituted T5 (calculatedM2H+m/z=1260.63) eluted at 39.1 minutes with an observed m/z of 1260.4(FIG. 12A). No peptide with m/z=1246+/−2 was observed in the trypticmass map of wt HSA at 39.1 minutes (FIG. 12B). The 39.1 min peak in (A)(m/z=1260.4) has an MS/MS spectrum consistent with the expectedpAF-substituted T5 peptide (see FIG. 13).

MS/MS of pAF-Containing HSA Polypeptide

MS/MS spectrum of pAF-T5 parent ion (m/z=1260.4). Singly-charged ionfragments were produced by collision-induced dissociation of thedoubly-charged parent ion. Peaks corresponding to predicted m/zintervals (y″ series) are indicated in the spectrum. The peak intervalsare consistent with the HSA T5 peptide containing a Cys34-pAFsubstitution. See FIG. 13.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a hApolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues 17, 34, 55, 56, 58, 60,81, 82, 86, 92, 94, 111, 114, 116, 119, 129, 170, 172, 173, 276, 277,280, 297, 300, 301, 313, 317, 321, 362, 363, 364, 365, 368, 375, 397,439, 442, 495, 496, 498, 500, 501, 505, 515, 538, 541, 542, 560, 562,564, 574, 581, identified according to the criteria of Example 1 (hA) isseparately substituted with a non-naturally encoded amino acid havingthe following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into hA are SEQ ID NO: 1.

The hA polypeptide variant comprising the carbonyl-containing amino acidis reacted with an aminooxy-containing PEG derivative of the form:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 3 and N is approximately 5,000 MW. Another PEGderivative that is conjugated to hA has a molecular weight of 30,000.The purified hA containing p-acetylphenylalanine dissolved at 10 mg/mLin 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-hA is then diluted into appropriate buffer for immediatepurification and analysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂

where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into hA polypeptides.

This example demonstrates a method for the generation of a hApolypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the followingresidues: E30, E74, Y103, K38, K41, K140, and K145. The hA polypeptideis prepared as described in Examples 1 and 2, except that the selectorcodon is introduced at two distinct sites within the nucleic acid.

Example 6

This example details conjugation of hA polypeptide to ahydrazide-containing PEG and subsequent in situ reduction.

A hA polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described in Examples 2 and 3. Oncemodified, a hydrazide-containing PEG having the following structure isconjugated to the hA polypeptide:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂

where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (C═O) group. Thepurified hA containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH₃ (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4 ° C. to RT for18-24 hours. Reactions are stopped by addition of 1 M Tris (SigmaChemical, St. Louis, Mo.) at about pH 7.6 to a final Tris concentrationof 50 mM or diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto a hA polypeptide and derivatization with mPEG-azide.

The following residues, 17, 34, 55, 56, 58, 60, 81, 82, 86, 92, 94, 111,114, 116, 119, 129, 170, 172, 173, 276, 277, 280, 297, 300, 301, 313,317, 321, 362, 363, 364, 365, 368, 375, 397, 439, 442, 495, 496, 498,500, 501, 505, 515, 538, 541, 542, 560, 562, 564, 574, 581, aresubstituted with the following non-naturally encoded amino acid (hA; SEQID NO: 1):

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into hA are SEQ ID NO: 2 (hA), SEQ ID NO: 3(muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), and 8, 9 or 10 described inExample 2 above. The hA polypeptide containing the propargyl tyrosine isexpressed in E. coli and purified using the conditions described inExample 3.

The purified hA containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3.

In this Example, the PEG will have the following structure:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃

where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid ina hA polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following positions ofhA: 17, 34, 55, 56, 58, 60, 81, 82, 86, 92, 94, 111, 114, 116, 119, 129,170, 172, 173, 276, 277, 280, 297, 300, 301, 313, 317, 321, 362, 363,364, 365, 368, 375, 397, 439, 442, 495, 496, 498, 500, 501, 505, 515,538, 541, 542, 560, 562, 564, 574, 581 (SEQ ID NO: 1), is substitutedwith the following non-naturally encoded amino acid as described inExample 7:

Once modified, a PEG is attached to the hA polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:

Me-PEG(N)—O—(CH₂)₂—N₃

and coupling procedures would follow those in Example 7. This willgenerate a hA polypeptide variant comprising a non-naturally encodedamino acid that is approximately isosteric with one of thenaturally-occurring, large hydrophobic amino acids and which is modifiedwith a PEG derivative at a distinct site within the polypeptide.

Example 9

This example details generation of a hA polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers.

The alkyne-containing hA polypeptide variant produced in Example 7 isreacted with a bifunctional PEG derivative of the form:

N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O-PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃

where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding hA polypeptide homodimer where the two hAmolecules are physically separated by PEG. In an analogous manner a hApolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to a hApolypeptide.

One residue of the following is substituted with the non-naturallyencoded amino acid below: 17, 34, 55, 56, 58, 60, 81, 82, 86, 92, 94,111, 114, 116, 119, 129, 170, 172, 173, 276, 277, 280, 297, 300, 301,313, 317, 321, 362, 363, 364, 365, 368, 375, 397, 439, 442, 495, 496,498, 500, 501, 505, 515, 538, 541, 542, 560, 562, 564, 574, 581 (hA, SEQID NO: 1) as described in Example 3.

Once modified, the hA polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The hA polypeptide variant (10mg/mL) and the aminooxy saccharide (21 mM) are mixed in aqueous 100 mMsodium acetate buffer (pH 5.5) and incubated at 37° C. for 7 to 26hours. A second saccharide is coupled to the first enzymatically byincubating the saccharide-conjugated hA polypeptide (5 mg/mL) withUDP-galactose (16 mM) and β-1,4-galacytosyltransferase (0.4 units/mL) in150 mM HEPES buffer (pH 7.4) for 48 hours at ambient temperature(Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11 Generation of a hA Polypeptide Homodimer, Heterodimer,Homomultimer, or Heteromultimer in Which the hA Molecules are LinkedDirectly

A hA polypeptide variant comprising the alkyne-containing amino acid canbe directly coupled to another hA polypeptide variant comprising theazido-containing amino acid, each of which comprise non-naturallyencoded amino acid substitutions at the sites described in, but notlimited to, Example 10. This will generate the corresponding hApolypeptide homodimer where the two hA polypeptide variants arephysically joined at the site II binding interface. In an analogousmanner a hA polypeptide polypeptide may be coupled to one or more otherpolypeptides to form heterodimers, homomultimers, or heteromultimers.Coupling, purification, and analyses are performed as in Examples 3, 6,and 7.

Example 12

This example describes conjugations of hA comprising a non-naturallyencoded amino acid with other biologically active molecules. The hAproduced in Example 2 herein is reacted with a desired biologicallyactive molecule such as a synthetic peptide, a small organic molecule, apolymer, a linker having one, two, three or more functional groupsavailable for coupling to hA or other biologically active molecules, aprotein or polypeptide other than hA, another hA molecule, orconjugation of a biologically active molecule to the non-natural aminoacid and another biologically active molecule attached to the cysteineat position 34 of SEQ ID NO: 1. The desired biologically active moleculeis reacted with the hA comprising a non-naturally encoded amino acidunder conditions that allow covalent bond formation between thefunctional group of the non-naturally encoded amino acid of the hA witha complementary functional group on the biologically active molecule.The covalently bonded hA and desired biologically active molecule arefurther purified if desired utilizing the methods known in the art ordescribed herein.

Example 13

This example details cloning and expression of a Fc with and without anon-naturally encoded amino acid in mammalian cells. [Cloning of the FcDNA into expression vector, transformation of mammalian cells] SEQ IDNO: 20 shows the wild-type Fc polynucleotide sequence. Thispolynucleotide sequence encodes a human IgG1-Fc with a signal sequencethat is absent in the purified protein. The codon underlined in thissequence was replaced by a selector codon, an amber codon, to obtain themutant protein. The codon underlined encodes the aspartic acid (Asp; D)amino acid which is the first amino acid of the mature protein. SEQ IDNO: 21 shows the polypeptide sequence of the Fc with the signalsequence. SEQ ID NO: 22 shows the sequence of the mature Fc protein. SEQID NO: 23 shows the polynucleotide sequence encoding the mature Fcprotein. See FIGS. 8A-D.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress Fc containing a non-naturally encoded amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into Fc, in response to an encoded selector codon.

Transient Production of WT and D1pAF-Fc

CHO-S Freestyle™ cells (passage 10; Invitrogen, Carlsbad, Calif.) wereseeded in 300 mL Freestyle™ CHO media (Invitrogen, Carlsbad, Calif.) at5×10⁵ cells/mL 24 hours prior to transfection. The transfection wasconducted according to the manufacturer's instructions. Briefly, 375 mLFreestyle MAX™ reagent (Invitrogen, Carlsbad, Calif.) was incubated withplasmid encoding wild-type (WT) Fc (415 μg) or plasmids encoding anorthogonal tRNA (SEQ ID NO: 24), an orthogonal tRNA synthetase (5 μg)(the encoding polynucleotide sequence shown as SEQ ID NO: 25) andFc-D1_(TAG) (140 μg) for 15 minutes. See FIGS. 9A and B for the tRNA andRS sequences. The non-naturally encoded amino acid will replace theaspartic acid (Asp; D) amino acid present at position 1 of the Fcsequence. The DNA transfection mix was added to 3.2×10⁸ cells in a totalof 300 mL. For cellular expression of protein from Fc-D1_(TAG),para-acetylphenylalanine (pAF) was added to a final concentration of 1mM. The cells were incubated at 34° C. at 8% CO₂ and 100 rpm. At 24hours post-transfection, Select Phytone UF hydrolysate (Becton,Dickinson and Company, Franklin Lakes, N.J.) was added to a finalconcentration of 0.1%. The cultures were harvested at 72 hourspost-transfection by centrifugation (10 minutes at 4,000 rpm), and thesupernatants were filter sterilized using a 0.22 μm filter. Purificationof WT and D1pAF-Fc from CHO-S culture supernatants

Clarified culture supernatants were loaded onto a 5 mL HiTrap rProteinAFF pre-packed column (GE Healthcare, Piscataway, N.J.). The column waswashed with 10 column volumes of PBS pH 7.4 prior to elution with 0.1Mglycine pH 3.0. Fractions containing Fc were neutralized toapproximately pH 7 with 0.03 volumes 0.5M Tris base. Fractions werepooled based on SDS-PAGE analysis, and the pool was concentrated with a10,000 molecular weight cut off spin apparatus (Amicon) and dialyzedoverni_(ght) against PBS pH 7.4+10% glycerol at 4° C. The Fcconcentration was determined using absorbance at 280 nm. The extinctioncoefficient used for the Fc with para-acetylphenylalanine incorporation,D1pAF, was 1.35. The extinction coefficient used for WT F^(c) was 1.40.N-terminal sequencing and mass spectrometry demonstrated properincorporation of pAF at the N-terminus of D1_(TAG). This protein isreferred to as D1pAF (Fc with pAF substituted at positi_(o)n 1).

Conjugation of 5K amino-oxy PEG to D1pAF-Fc

Purified WT-Fc and D1pAF were buffer exchanged into conjugation buffer(20 mM sodium acetate, 20 g/L glycine, 5 g/L mannitol, 1 mM EDTA, pH4.0) and concentrated to approximately 1.5 mg/mL. The samples wereincubated in conjugation buffer alone or with 5K amino-oxypoly(ethylene)-glycol (PEG) at a final concentration of 15 g/L (50-foldmolar excess over Fc monomer). Acetic hydrazide was added to allreactions at a final concentration of 50 mM to catalyze the conjugationreaction. Reactions were allowed to proceed at 28° C. for 48 hours. A 50aliquot was removed at 24 hours. Three μg of each sample was analyzed bySDS PAGE. 4-12% bis-tris SDS-PAGE was performed under reducing andnon-reducing conditions and coomassie stained. Reduced samples wereseparated using an MES buffer system, and non-reduced samples wereseparated with a MOPS buffer system.

FIG. 7 shows 5K PEG conjugation to the amino terminal pAF residue ofhuman IgG 1-Fc. Purified Fc (WT) and D1pAF-substituted Fc (D1pAF) wereincubated in the presence (+) or absence (−) of 5K amino-oxypoly(ethylene)-glycol (PEG) for 24 or 48 hours at 28° C. Reduced (FIG.7A) or non-reduced (FIG. 7B) samples were analyzed by coomassie bluestaining of protein separated by SDS-PAGE.

FIG. 7A shows D1pAF+PEG exhibited a mass shift compared to D1pAFincubated without PEG (lanes 7 vs. 5, respectively). In contrast, nomass shift was observed after WT Fc was incubated with 5K PEG (lanes 3and 6), demonstrating that the mass shift is pAF-dependent. SDS-PAGEanalysis of non-reduced samples demonstrated that intact-Fc dimers wereconjugated on one (PEG-D1pAF+D1pAF) or two (PEG-D1pAF+PEG-D1pAF) arms ofthe molecule (FIG. 7B). Staining intensity indicated that the majorityof the material was doubly PEGylated Fc dimer (lane 7). The lower bandspresent in panel (FIG. 7B) are reduced forms of PEGylated andunPEGylated Fc monomer.

As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the description discussedadding macromolecular polymers to a Fc comprising a non-naturallyencoded amino acid with the understanding that the compositions,methods, techniques and strategies described thereto are also applicable(with appropriate modifications, if necessary and for which one of skillin the art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above and/orto other Fc molecules.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to those of ordinary skill in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

SEQ ID NO SEQUENCE Notes 3CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCGCTGG M. jannaschiitRNA TTCAAATCCGGCCCGCCGGACCA mtRNA_(CUA) ^(Tyr) 4 CCCAGGGTAG CCAAGCTCGGCCAACGGCGAC GGACTCTAA HLAD03; an tRNA ATCCGTTCTC GTAGGAGTTC GAGGGTTCGAATCCCTTCCC TGGGACCA optimized amber supressor tRNA 5 GCGAGGGTAGCCAAGCTCGG CCAACGGCGA CGGACTTCCT AATCCGTTCT HL325A: an optimized tRNACGTAGGAGTT CGAGGGTTCG AATCCCTCCC CTCGCACCA AGGA frameshift supressortRNA 6 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMID AminoacyltRNA RS LQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFsynthetase for theQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTYYYL incorporation ofp- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-L-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY p-Az-PheRS (6)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 7MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTSHYL incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVD benzoyl-L-DSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY phenylalanineEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL p-BpaRS(1) 8 MDEFE MIKRN TSEIISEEEL REVLK KDEKS AAIGF EPSGK IHLGH Aminoacyl tRNA RS YLQIK KMIDL QNAGFDIIIL LADLH AYLNQ KGELD EIRKI GDYNK synthetase for the KVFEA MGLKA KYVYGSPFQL DKDYT LNVYR LALKT TLKRA RRSME incorporation of LIARE DENPK VAEVIYPIMQ VNAIY LAVD VAVGG MEQRK IHMLA propargyl- RELLP KKVVC IHNPV LTGLDGEGKM SSSKG NFIAV DDSPE EIRAK phenylalanine IKKAY CPAGV VEGNP IMEIAKYFLE YPLTI KRPEK FGGDL TVNSY Propargyl-PheRS EELES LFKNK ELHPM DLKNAVAEEL IKILE PIRKR L 9 MDEFE MIKRN TSEII SEEEL REVLK KDEKS AAIGF EPSGKIHLGH Aminoacyl tRNA RS YLQIK KMIDL QNAGF DIIIL LADLH AYLNQ KGELD EIRKIGDYNK synthetase for the KVFEA MGLKA KYVYG SPFQL DKDYT LNVYR LALKT TLKRARRSME incorporation of LIARE DENPK VAEVI YPIMQ VNIPY LPVD VAVGG MEQRKIHMLA propargyl- RELLP KKVVC IHNPV LTGLD GEGKM SSSKG NFIAV DDSPE EIRAKphenylalanine IKKAY CPAGV VEGNP IMEIA KYFLE YPLTI KRPEK FGGDL TVNSYPropargyl-PheRS EELES LFKNK ELHPM DLKNA VAEEL IKILE PIRKR L 10 MDEFEMIKRN TSEII SEEEL REVLK KDEKS AAIGF EPSGK IHLGH Aminoacyl tRNA RS YLQIKKMIDL QNAGF DIIIL LADLH AYLNQ KGELD EIRKI GDYNK synthetase for the KVFEAMGLKA KYVYG SKFQL DKDYT LNVYR LALKT TLKRA RRSME incorporation of LIAREDENPK VAEVI YPIMQ VNAIY LAVD VAVGG MEQRK IHMLA propargyl- RELLP KKVVCIHNPV LTGLD GEGKM SSSKG NFIAV DDSPE EIRAK phenylalanine IKKAY CPAGVVEGNP IMEIA KYFLE YPLTI KRPEK FGGDL TVNSY Propargyl-PheRS EELES LFKNKELHPM DLKNA VAEEL IKILE PIRKR L 11MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSNF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYQ incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY p-Az-PheRS(1)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 12MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYQ incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY p-Az-PheRS(3)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 13MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPVHYQ incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY p-Az-PheRS(4)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 14MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPSHYQ incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY p-Az-PheRS(2)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 15MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGCHYR incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDacetyl-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY (LW1)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 16MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGTHYR incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDacetyl-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY (LW5)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 17MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGGHYL incorporationof p- GVDVIVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDacetyl-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY (LW6)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 18MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSRF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNVIHYD incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY (AzPheRS-5)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 19MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMID Aminoacyl tRNA RSLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTF synthetase forthe QLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTYYYL incorporationof p- GVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDazido-phenylalanineDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSY (AzPheRS-6)EELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL

1. An hPP or hA polypeptide comprising one or more non-naturally encodedamino acids.
 2. The hPP or hA polypeptide of claim 1, wherein the hPP orhA polypeptide comprises one or more post-translational modifications.3. The hPP or hA polypeptide of claim 1, wherein the polypeptide islinked to a linker, polymer, or biologically active molecule.
 4. The hPPor hA polypeptide of claim 3, wherein the polypeptide is linked to awater soluble polymer.
 5. The hPP or hA polypeptide of claim 1, whereinthe polypeptide is linked to a bifunctional polymer, multifunctionalpolymer, bifunctional linker, multifunctional linker or at least onebiologically active molecule.
 6. The hPP or hA polypeptide of claim 5,wherein the linker or polymer is linked to a second polypeptide.
 7. ThehPP or hA polypeptide of claim 6, wherein the second polypeptide is abiologically active molecule.
 8. The hPP or hA polypeptide of claim 4,wherein the water soluble polymer comprises a poly(ethylene glycol)moiety.
 9. The hPP or hA polypeptide of claim 4, wherein said watersoluble polymer is linked to a non-naturally encoded amino acid presentin said hPP or hA polypeptide.
 10. The hA polypeptide of claim 1,wherein the non-naturally encoded amino acid is substituted at aposition selected from the group consisting of residues 17, 34, 55, 56,58, 60, 81, 82, 86, 92, 94, 111, 114, 116, 119, 129, 170, 172, 173, 276,277, 280, 297, 300, 301, 313, 317, 321, 362, 363, 364, 365, 368, 375,397, 439, 442, 495, 496, 498, 500, 501, 505, 515, 538, 541, 542, 560,562, 564, 574, 581 from SEQ ID NO:
 1. 12. The hPP or hA polypeptide ofclaim 1, wherein the hPP or hA polypeptide comprises one or more aminoacid substitution, addition or deletion that modulates at least onebiological activity of the hPP or hA
 13. The hPP or hA polypeptide ofclaim 1, wherein the hPP or hA polypeptide comprises one or more aminoacid substitution, addition or deletion that increases the stability orsolubility of the hPP or hA polypeptide.
 14. The hPP or hA polypeptideof claim 1, wherein the hPP or hA polypeptide comprises one or moreamino acid substitution, addition or deletion that increases theexpression of the hPP or hA polypeptide in a recombinant host cell orsynthesized in vitro.
 15. The hPP or hA polypeptide of claim 1, whereinthe hPP or hA polypeptide comprises one or more amino acid substitution,addition or deletion that increases protease resistance of the hPP or hApolypeptide.
 16. The hPP or hA polypeptide of claim 1, wherein thenon-naturally encoded amino acid is reactive toward a linker, polymer,or biologically active molecule that is otherwise unreactive toward anyof the 20 common amino acids in the polypeptide.
 17. The hPP or hApolypeptide of claim 1, wherein the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.
 18. The hPP or hA polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a carbonyl group.
 19. The hPPor hA polypeptide of claim 17, wherein the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R3 is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R4 is H, an amino acid, a polypeptide,or a carboxy terminus modification group.
 20. The hPP or hA polypeptideof claim 17, wherein the non-naturally encoded amino acid comprises anaminooxy group.
 21. The hPP or hA polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a hydrazide group.
 22. ThehPP or hA polypeptide of claim 17, wherein the non-naturally encodedamino acid comprises a hydrazine group.
 23. The hPP or hA polypeptide ofclaim 17, wherein the non-naturally encoded amino acid residue comprisesa semicarbazide group.
 24. The hPP or hA polypeptide of claim 17,wherein the non-naturally encoded amino acid residue comprises an azidegroup.
 25. The hPP or hA polypeptide of claim 24, wherein thenon-naturally encoded amino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.
 26. The hPP or hA polypeptide of claim 17, whereinthe non-naturally encoded amino acid comprises an alkyne group.
 27. ThehPP or hA polypeptide of claim 26, wherein the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.
 28. The hPP or hA polypeptide of claim 4, whereinthe water soluble polymer has a molecular weight of between about 0.1kDa and about 100 kDa.
 29. The hPP or hA polypeptide of claim 28,wherein the water soluble polymer has a molecular weight of betweenabout 0.1 kDa and about 50 kDa.
 30. The hPP or hA polypeptide of claim4, which is made by reacting an hPP or hA polypeptide comprising acarbonyl-containing amino acid with a water soluble polymer comprisingan aminooxy, hydrazine, hydrazide or semicarbazide group.
 31. The hPP orhA polypeptide of claim 30, wherein the aminooxy, hydrazine, hydrazideor semicarbazide group is linked to the water soluble polymer through anamide linkage.
 32. The hPP or hA polypeptide of claim 4, which is madeby reacting a water soluble polymer comprising a carbonyl group with apolypeptide comprising a non-naturally encoded amino acid that comprisesan aminooxy, a hydrazine, a hydrazide or a semicarbazide group.
 33. ThehPP or hA polypeptide of claim 4, which is made by reacting an hPP or hApolypeptide comprising an alkyne-containing amino acid with a watersoluble polymer comprising an azide moiety.
 34. The hPP or hApolypeptide of claim 4, which is made by reacting an hPP or hApolypeptide comprising an azide-containing amino acid with a watersoluble polymer comprising an alkyne moiety.
 35. The hPP or hApolypeptide of claim 17, wherein the azide or alkyne group is linked toa water soluble polymer through an amide linkage.
 36. The hPP or hApolypeptide of claim 4, wherein the water soluble polymer is a branchedor multiarmed polymer.
 37. The hPP or hA polypeptide of claim 36,wherein each branch of the water soluble polymer has a molecular weightof between about 1 kDa and about 100 kDa.
 38. The hPP or hA polypeptideof claim 1, wherein the non-naturally encoded amino acid comprises asaccharide moiety.
 39. The hPP or hA polypeptide of claim 3, wherein thelinker, polymer, or biologically active molecule is linked to thepolypeptide via a saccharide moiety.
 40. An isolated polynucleotidemolecule that encodes the amino acid sequence set forth in SEQIDNO:1comprising a selector codon, wherein the selector codon is selected fromthe group consisting of an amber codon, ochre codon, opal codon, aunique codon, a rare codon, and a four-base codon.
 41. A method ofmaking the hPP or hA polypeptide of claim 3, the method comprisingcontacting an isolated hPP or hA polypeptide comprising a non-naturallyencoded amino acid with a linker, polymer, or biologically activemolecule comprising a moiety that reacts with the non-naturally encodedamino acid.
 42. The method of claim 41, wherein the polymer comprises amoiety selected from a group consisting of a water soluble polymer andpoly(ethylene glycol).
 43. The method of claim 41, wherein thenon-naturally encoded amino acid comprises a carbonyl group, an aminooxygroup, a hydrazide group, a hydrazine group, a semicarbazide group, anazide group, or an alkyne group.
 44. The method of claim 41, wherein thenon-naturally encoded amino acid comprises a carbonyl moiety and thelinker, polymer, or biologically active molecule comprises an aminooxy,a hydrazine, a hydrazide or a semicarbazide moiety.
 45. The method ofclaim 44, wherein the aminooxy, hydrazine, hydrazide or semicarbazidemoiety is linked to the linker, polymer, or biologically active moleculethrough an amide linkage.
 46. The method of claim 41, wherein thenon-naturally encoded amino acid comprises an alkyne moiety and thelinker, polymer, or biologically active molecule comprises an azidemoiety.
 47. The method of claim 41, wherein the non-naturally encodedamino acid comprises an azide moiety and the linker, polymer, orbiologically active molecule comprises an alkyne moiety.
 48. The methodof claim 42, wherein the azide or alkyne moiety is linked to a linker,polymer, or biologically active molecule through an amide linkage. 49.The method of claim 42, wherein the poly(ethylene glycol) moiety has anaverage molecular weight of between about 0.1 kDa and about 100 kDa. 50.The method of claim 42, wherein the poly(ethylene glycol) moiety is abranched or multiarmed polymer.
 51. A composition comprising the hPP orhA polypeptide of claim 1 and a pharmaceutically acceptable carrier. 52.The composition of claim 51, wherein the non-naturally encoded aminoacid is linked to a water soluble polymer.
 53. A method of treating apatient having a disorder modulated by hPP or hA comprisingadministering to the patient a therapeutically-effective amount of thecomposition of claim
 51. 54. A cell comprising the nucleic acid of claim40.
 55. The cell of claim 54, wherein the cell comprises an orthogonaltRNA synthetase or an orthogonal tRNA.
 56. A method of making an hPP orhA polypeptide comprising a non-naturally encoded amino acid, the methodcomprising, culturing cells comprising a polynucleotide orpolynucleotides encoding an hPP or hA polypeptide comprising a selectorcodon, an orthogonal RNA synthetase and an orthogonal tRNA underconditions to permit expression of the hPP or hA polypeptide comprisinga non-naturally encoded amino acid; and purifying the hPP or hApolypeptide.
 57. A method of modulating serum half-life or circulationtime of an hPP or hA polypeptide, the method comprising substituting oneor more non-naturally encoded amino acids for any one or more naturallyoccurring amino acids in the hPP or hA polypeptide.
 58. An hPP or hApolypeptide encoded by a polynucleotide having a sequence shown in SEQID NO: 1, wherein said polynucleotide comprises a selector codon, andwherein said polypeptide comprises at least one non-naturally encodedamino acid.
 59. The hPP or hA polypeptide of claim 58, wherein thenon-naturally encoded amino acid is linked to a linker, polymer, watersoluble polymer, or biologically active molecule.
 60. The hPP or hApolypeptide of claim 59, wherein the water soluble polymer comprises apoly(ethylene glycol) moiety.
 61. The hA polypeptide of claim 58,wherein the non-naturally encoded amino acid is substituted at aposition selected from the group consisting of residues 17, 34, 55, 56,58, 60, 81, 82, 86, 92, 94, 111, 114, 116, 119, 129, 170, 172, 173, 276,277, 280, 297, 300, 301, 313, 317, 321, 362, 363, 364, 365, 368, 375,397, 439, 442, 495, 496, 498, 500, 501, 505, 515, 538, 541, 542, 560,562, 564, 574, 581, and any combination thereof from SEQ ID NO:
 1. 62.The hPP or hA polypeptide of claim 58, wherein the non-naturally encodedamino acid comprises a carbonyl group, an aminooxy group, a hydrazidegroup, a hydrazine group, a semicarbazide group, an azide group, or analkyne group.
 63. The hPP or hA polypeptide of claim 60, wherein thepoly(ethylene glycol) moiety has a molecular weight of between about 0.1kDa and about 100 kDa.
 64. The hPP or hA polypeptide of claim 60,wherein the poly(ethylene glycol) moiety is a branched or multiarmedpolymer.
 65. The hPP or hA polypeptide of claim 64, wherein thepoly(ethylene glycol) moiety has a molecular weight of between about 1kDa and about 100 kDa.
 66. A composition comprising the hPP or hApolypeptide of claim 58 and a pharmaceutically acceptable carrier. 67.An hPP or hA polypeptide comprising one or more amino acid substitution,addition or deletion that increases the expression of the hPP or hApolypeptide in a recombinant host cell.
 68. An hPP or hA polypeptidecomprising one or more non-naturally encoded amino acids, wherein saidnon-naturally encoded amino acid is bonded to a molecule selected fromthe group consisting of small organic molecules, chemically synthesizedpeptides, ribosomally synthesized polypeptides, polymers, and linkers.69. An HPP or hA polypeptide comprising a water soluble polymer linkedby a covalent bond to the hPP or hA polypeptide at a single amino acid.70. The hPP or hA polypeptide of claim 69, wherein the water solublepolymer comprises a poly(ethylene glycol) moiety.
 71. The hPP or hApolypeptide of claim 69, wherein the amino acid covalently linked to thewater soluble polymer is a non-naturally encoded amino acid.
 72. The hPPor hA polypeptide of claim 71, wherein the non-naturally encoded aminoacid is substituted at a position selected from the group 17, 34, 55,56, 58, 60, 81, 82, 86, 92, 94, 111, 114, 116, 119, 129, 170, 172, 173,276, 277, 280, 297, 300, 301, 313, 317, 321, 362, 363, 364, 365, 368,375, 397, 439, 442, 495, 496, 498, 500, 501, 505, 515, 538, 541, 542,560, 562, 564, 574, 581 corresponding to SEQ ID NO:
 1. 73. An hPP or hApolypeptide comprising at least one linker, polymer, or biologicallyactive molecule, wherein said linker, polymer, or biologically activemolecule is attached to the polypeptide through a functional group of anon-naturally encoded amino acid ribosomally incorporated into thepolypeptide.
 74. The hPP or hA polypeptide of claim 1, wherein the hPPor hA polypeptide comprises one or more amino acid substitution,addition, or deletion that modulates immunogenicity of the hPP or hApolypeptide.
 75. The hPP or hA polypeptide of claim 1, wherein the hPPor hA polypeptide comprises one or more amino acid substitution,addition, or deletion that modulates serum half-life or circulation timeof a biologically active molecule.
 76. A method of modulatingimmunogenicity of a biologically active molecule, the method comprisingsubstituting one or more non-naturally encoded amino acids for any oneor more naturally occurring amino acids in the hPP or hA polypeptide.